Oligodeoxynucleotide and its use to induce an immune response

ABSTRACT

D type CpG oligodeoxynucleotides are provided herein that include a sequence represented by the following formula: 
       5′X 1 X 2 X 3 Pu 1 Py 2 CpGPu 3 Py 4 X 4 X 5 X 6 (W) M (G) N -3′
 
     wherein the central CpG motif is unmethylated, Pu is a purine nucleotide, Py is a pyrimidine nucleotide, X and W are any nucleotide, M is any integer from 0 to 10, and N is any integer from 4 to 10. Methods of using these oligodeoxynucleotides to induce an immune response are provided.

REFERENCE TO RELATED APPLICATIONS

This is application is a continuation of U.S. patent application Ser.No. 11/131,672, filed May 17, 2005, which is a continuation of U.S.patent application Ser. No. 10/068,160, filed Feb. 6, 2002, which is acontinuation-in-part of U.S. patent application Ser. No. 09/958,713,filed on Oct. 7, 2002, which is the United States national phaseapplication under 35 U.S.C. §371 of PCT Application No. PCT/US00/09839,filed on Apr. 12, 2000, which claims priority to U.S. Provisional PatentApplication No. 60/128,898, filed on Apr. 12, 1999, all of which areincorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was supported in part by Military InterdepartmentalPurchase Request MM8926. The Government of the United States has certainrights in this invention.

FIELD OF THE DISCLOSURE

The present invention relates to the induction of an immune response,specifically to oligodeoxynucleotides including a CpG motif and theiruse in inducing an immune response.

BACKGROUND

Cells of the immune system recognize and are activated by conservedpathogen associated molecular patterns (PAMPs) in infectious agents. Theunmethylated CpG dimers embedded in bacterial DNA, as well as certainsynthetic oligodeoxynucleotides (ODNs) containing unmethylated CpGsequences (termed a CpG motif) that emulated them, are more frequent inthe genomes of bacteria and viruses than vertebrates. Recent studiessuggest that immune recognition of these motifs may contribute to thehost's innate immune response (Klinman et al., Proc. Natl. Acad. Sci.USA 93: 2879, 1996; Yi et al, J. Immun. 157: 5394, 1996; Liang et al.,J. Clin. Invest. 98:1119, 1996; Krieg et al., 374 Nature 374: 546,1995).

In mice, CpG DNA induces proliferation in almost all (>95%) of B cellsand increases immunoglobulin (Ig) secretion. This B-cell activation byCpG DNA is T-cell independent and antigen non-specific. In addition toits direct effects on B cells, CpG DNA has also been shown to activatecells of the immune system (see, for example, International PatentApplications WO 95/26204, WO 96/02555, WO 98/11211, WO 98/18810, WO98/37919, WO 98/40100, WO 98/52581, PCT/US98/047703, and PCT/US99/07335;U.S. Pat. No. 5,663,153).

Although bacterial DNA and certain oligonucleotides can induce a murineimmune response, little is known about the immunostimulatory capacity ofthese materials for the human immune system (Ballas et al., 157 J.Immun. 157: 1840 1996). In addition, differences in the responsivenessof human and murine B cells to certain stimuli render it difficult toextrapolate results obtained from mouse to man.

In view of the above, there exists a need for oligonucleotides thatinduce an immune response in humans. In addition, there is a need formethods utilizing CpG containing oligonucleotides in the treatment ofhuman diseases.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file[4239-61997-03_Sequence_Listing.txt, May 9, 2011, 23.1 KB], which isincorporated by reference herein.

BRIEF SUMMARY OF SPECIFIC EMBODIMENTS

A substantially pure or isolated oligodeoxynucleotide (ODN) is disclosedherein that is at least about 16 nucleotides in length. The ODNs arereferred to herein as D type ODNs, and are distinct from the previouslydescribed K type ODNs.

The oligodeoxynucleotide includes a sequence represented by thefollowing formula:

5′X₁X₂X₃Pu₁Py₂CpGPu₃Py₄X₄X₅X₆(W)_(M)(G)_(N)-3′

wherein the central CpG motif is unmethylated, Pu is a purinenucleotide, Py is a pyrimidine nucleotide, X and W are any nucleotide, Mis any integer from 0 to 10, and N is any integer from 4 to 10. In oneembodiment, X₁X₂X₃ and X₄X₅X₆ are self-complementary. In anotherembodiment, at least two G's are included at the 5′ end of the molecule,such that the oligodeoxynucleotide includes a sequence represented bythe formula:

5′GGX₁X₂X₃Pu₁Py₂CpGPu₃Py₄X₄X₅X₆(W)_(M)(G)_(N)-3′

These oligodeoxynucleotides are also referred to as “D type”oligodeoxynucleotides. In one embodiment, Pu₁ Py₂ CpG Pu₃ Py₄ arephosphodiester bases. In one specific, non-limiting example, Pu₁ is anadenine and Py₂ is a tyrosine. In another specific, non-limitingexample, Pu₃ is an adenine and Py₄ is a tyrosine.

These oligodeoxynucleotides stimulate cell types of the immune system tomount distinct immune responses. In one embodiment, the oligonucleotideinduces production of a cytokine. Specific, non-limiting examples areinterferon-gamma (IFN-γ), interferon-alpha (IFN-α), IP-10, or IL-10. Inanother embodiment, a method is provided for activating a cell of theimmune system. These cells include, but are not limited to, dendriticcells, natural killer (NK cells), and monocytes. Thus by employing Dtype ODN, the immune system can be manipulated to support specifictherapeutic goals.

Also disclosed herein is a delivery complex for D typeoligodeoxynucleotides and pharmacological composition comprising theD-type oligodeoxynucleotides.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a set of bar graphs illustrating the response of PBMC to K andD ODN. PBMC from 35 donors were stimulated for 72 h with K or D ODN (3mM). D19 (GGTGCATCGATGCAGGGGGG, SEQ ID NO: 1) and D29(GGTGCACCGGTGCAGGGGGG, SEQ ID NO: 2) exemplify the response of PBMC toODN that selectively induce IFN-γ, whereas K3 (ATCGACTCTCGAGCGTTCTC, SEQID NO: 3) and K23 (TCGAGCGTTCT, SEQ ID NO: 4) exemplify ODN that induceIgM and IL-6 secretion and cell proliferation but little IFN-γ. ControlODN have the CG dimer reversed or the C in the CG dimer replaced by a T;therefore, control D (GGTGCACGCGTGCAGGGGGG, SEQ ID NO: 5) and control K(TGCAGGCTTCTC, SEQ ID NO: 6). Bases in bold-face type arephosphorodiester, and those in normal type are phosphorothioate.Cytokine and immunoglobulin (Ig) concentrations in supernatants weredetermined by ELISA, and cell proliferation was assessed by [³H]thymidine uptake. All assays were done in triplicate. Statisticalsignificance was determined by the nonparametric Mann-Whitney U test.

FIG. 2 is several sets of sequences and bar graphs showing theparameters governing D ODN induced immune activation. FIG. 2A is a setof sequences and a bar graph demonstrating that D ODNs require anunmethylated CpG. FIG. 2B is a set of sequences and a bar graphdemonstrating that D ODNs require a phosphodiester backbone for optimalstimulation. FIG. 2C is a set of sequences and a bar graph demonstratingthat a self complementary Pu₁Py₂ CpG Pu₃Py₄ increases the stimulationindex. FIG. 2D is a set of sequences and a bar graph showing how size ofan ODN affects stimulation index. FIG. 2E is a set of sequences and abar graph demonstrating that increased stimulation is obtained with aself complementary flanking region. FIG. 2F is a set of sequences and abar graph demonstrating that 3′ poly-G sequences can be used to improveactivity of D ODNs. The ODN shown here are representative of 120 ODNused to characterize the structural requirements of D ODN. CGs areunderlined; immunostimulatory motifs are in bold; extended motifs are initalics; methylated bases have an asterisk; dots indicate identity; andshaded backgrounds identify phosphorodiester-linked bases. Importantbase changes in the sequence are circled. Data is expressed asstimulation indices, representing the fold increase in cytokinesecretion relative to unstimulated cells from the same donor. Barsrepresent the mean and SE of 20 different experiments. The ODN shown donot induce significant levels of IgM or proliferation. Statisticalsignificance was determined by the non-parametric Mann-Whitney U ornonparametric ANOVA: *, p <0.05; **, p<0.01; ***, p<0.001.

FIG. 3 shows the cell type-dependent response to CpG ODN. Fold increasein IFN-γ production by NK-92 cells and IL-6-secreting cell number byRPMI 8226 cells in response to ODN (3 mM for NK cells and 1 mM for Bcells). The number of cells secreting IL-6 was determined by ELISPOT,and the secretion of IFN-g was determined at 72 h in culturesupernatants by ELISA. Sequences: D19, D29 (see FIG. 1), D28(GGTGCGTCGATGCAGGGGGG, SEQ ID NO: 7), K16 (TC-GACTCTCGAGCGTTCTC, SEQ IDNO: 8), K19 (ACTCTCGAGCGTTCTC, SEQ ID NO:9), K101 (CTCGAGCGTTCT, SEQ IDNO: 10). Statistical significance determined by non-parametricMann-Whitney U and non-parametric ANOVA tests.

FIGS. 4A-F show the results of a study where PBMC from 8-20 normal humandonors (A, C and E) and 20 rhesus macaques (B, D and F) were stimulatedfor 72 hours with a panel of “K”, “D” or control ODN (3 μM). IL-6 (E andF) and IFN-α (A and B) levels in culture supernatants were determined byELISA while cell proliferation was assessed by [H]³ thymidine uptake (Cand D). Note that D ODN induce the secretion of IFNα while K ODN inducecell proliferation and IL-6 production. All assays were performed intriplicate. Statistical significance was determined by ANOVA of lognormalized data. * p<0.05; ** p<0.01.

FIGS. 5A-C shows the results of a study where PBMC from rhesus macaques(N=12-20) were stimulated in vitro for 72 hours with a mixture of D19,D29 and D35 (1 μM each) or K3 and K123 (1.5 μM each). D122 and K163 wereused in the control ODN mixture. Levels of IL-6 (B) and IFNα (C) inculture supernatants were measured by ELISA, while proliferation wasmeasured by [H]³-thymidine uptake (A). Statistical significance wasdetermined by ANOVA of the normalized data. ** p<0.01.

FIG. 6 shows the results of a study where Macaques (3/group) wereimmunized on day 0 s.c. with 4 μg of OVA plus 125 μg of alum and 250 μgof a mixture of (D19+D29), (K3+K23) or control (AA3M) ODN. Monkeys wereboosted 12 weeks later (black arrow). Serum IgG anti-OVA titers weredetermined by ELISA. Values represent the geometric mean titer±SEM. Theanti-OVA IgG titers in the group that received “D” ODN are significantlyhigher (p<0.01).

FIG. 7 shows the results of a study were Rhesus macaques were primedsubcutaneously (s.c.) with 250 μg of alum-adjuvanted HKLV alone (N=6) orcombined with 500 μg of a mixture of (D19, D29 and D35) (N=5) or (K3 andK123) (N=5) and boosted 4 weeks later. On week 14, the monkeys werechallenged on the opposite forehead with 10⁷ metacyclic promastigotes.The average size of the resultant lesions is shown as the mean area(calculated as mean diameter/2)²×pi). Macaques immunized withHKLV-ALUM-“D”ODN had significantly smaller lesions (p<0.01).

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS I. Abbreviations

A: adenine

Ab: antibody

APC: antigen presenting cell

C: cytosine

APC: antigen presenting cell

CpG ODN: an oligodeoxynucleotide (either a D or a K type) including aCpG motif.

DC: dendritic cell

FCS: fetal calf serum

G: guanine

h: hour

HKLV: heat-killed leishmania vaccine

IFN-α: interferon alpha

IFN-γ: interferon gamma

IL-10: interleukin 10

mm: millimeter

mRNA: messenger ribonucleic acid.

ODN: oligodeoxynucleotide

Pu: purine

Py: pyrimidine

s.c.: subcutaneous

T: thymine

μg: microgram

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. The term“comprises” means “includes.” It is further to be understood that allbase sizes or amino acid sizes, and all molecular weight or molecularmass values, given for nucleic acids or polypeptides are approximate,and are provided for description. Although methods and materials similaror equivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials aredescribed below. In case of conflict, the present specification,including explanations of terms, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

Allergen: A substance that can induce an allergic or asthmatic responsein a susceptible subject. The list of allergens is enormous and caninclude pollens, insect venoms, animal dander dust, fungal spores anddrugs (e.g. penicillin). Examples of natural, animal and plant allergensinclude proteins specific to the following genera: Canine (Canisfamiliaris); Dermatophagoides (e.g. Dermatophagoides farinae); Felis(Felis domesticus); Ambrosia (Ambrosia artemiisfolia); Lolium (e.g.Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeriajaponica); Alternaria (Alternaria alternata); Alder; Alnus (Alnusgultinosa); Betula (Betula verrucosa); Quercus (Quercus alba); Olea(Olea europa); Artemisia (Artemisia vulgaris); Plantago (e.g. Plantagolanceolata); Parietaria (e.g. Parietaria officinalis or Parietariajudaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apismultiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressusarizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperussabinoides, Juniperus virginiana, Juniperus communis and Juniperusashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g. Chamaecyparisobtusa); Periplaneta (e.g. Periplaneta americana); Agropyron (e.g.Agropyron repens); Secale (e.g. Secale cereale); Triticum (e.g. Triticumaestivum); Dactylis (e.g. Dactylis glomerata); Festuca (e.g. Festucaelatior); Poa (e.g. Poa pratensis or Poa compressa); Avena (e.g. Avenasativa); Holcus (e.g. Holcus lanatus); Anthoxanthum (e.g. Anthoxanthumodoratum); Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g.Agrostis alba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalarisarundinacea); Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghumhalepensis); and Bromus (e.g. Bromus inermis). The term “allergy” refersto acquired hypersensitivity to a substance (allergen). An “allergicreaction” is the response of an immune system to an allegen in a subjectallergic to the allergen. Allergic conditions include eczema, allergicrhinitis or coryza, hay fever, bronchial asthma, urticaria (hives) andfood allergies, and other atopic conditions.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes.

Anti-infectious agent: A substance (such as a chemical compound,protein, antisense oligonucleotide, or other molecule) of use intreating infection of a subject. Anti-infectious agents include, but arenot limited to, anti-fungals, anti-virals, and antibiotics.

Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA) has twostrands, a 5′->3′ strand, referred to as the plus strand, and a 3′->5′strand (the reverse compliment), referred to as the minus strand.Because RNA polymerase adds nucleic acids in a 5′->3′ direction, theminus strand of the DNA serves as the template for the RNA duringtranscription. Thus, the RNA formed will have a sequence complementaryto the minus strand and identical to the plus strand (except that U issubstituted for T).

Antisense molecules are molecules that are specifically hybridizable orspecifically complementary to either RNA or the plus strand of DNA.Sense molecules are molecules that are specifically hybridizable orspecifically complementary to the minus strand of DNA. Antigenemolecules are either antisense or sense molecules directed to a dsDNAtarget. In one embodiment, an antisense molecule specifically hybridizesto a target mRNA and inhibits transcription of the target mRNA.

Asthma: A disorder of the respiratory system characterized byinflammation, narrowing of the airways and increased reactivity of theairways to inhaled agents. Asthma is frequently, although notexclusively associated with atopic or allergic symptoms.

Autoimmune disorder: A disorder in which the immune system produces animmune response (e.g. a B cell or a T cell response) against anendogenous antigen, with consequent injury to tissues.

CpG or CpG motif: A nucleic acid having a cytosine followed by a guaninelinked by a phosphate bond in which the pyrimidine ring of the cytosineis unmethylated. The term “methylated CpG” refers to the methylation ofthe cytosine on the pyrimidine ring, usually occurring the 5-position ofthe pyrimidine ring. A CpG motif is a pattern of bases that include anunmethylated central CpG surrounded by at least one base flanking (onthe 3′ and the 5′ side of) the central CpG. Without being bound bytheory, the bases flanking the CpG confer part of the activity to theCpG oligodeoxynucleotide. A CpG oligonucleotide is an oligonucleotidethat is at least about ten nucleotides in length and includes anunmethylated CpG. CpG oligonucleotides include both D and K typeoligodeoxynucleotides (see below). CpG oligodeoxynucleotides aresingle-stranded. The entire CpG oligodeoxynucleotide can be unmethylatedor portions may be unmethylated. In one embodiment, at least the C ofthe 5′ CG 3′ is unmethylated.

Cancer: A malignant neoplasm that has undergone characteristic anaplasiawith loss of differentiation, increase rate of growth, invasion ofsurrounding tissue, and is capable of metastasis. For example, thyroidcancer is a malignant neoplasm that arises in or from thyroid tissue,and breast cancer is a malignant neoplasm that arises in or from breasttissue (such as a ductal carcinoma). Residual cancer is cancer thatremains in a subject after any form of treatment given to the subject toreduce or eradicate thyroid cancer. Metastatic cancer is a cancer at oneor more sites in the body other than the site of origin of the original(primary) cancer from which the metastatic cancer is derived.

Chemotherapy; chemotherapeutic agents: As used herein, any chemicalagent with therapeutic usefulness in the treatment of diseasescharacterized by abnormal cell growth. Such diseases include tumors,neoplasms, and cancer as well as diseases characterized by hyperplasticgrowth such as psoriasis. In one embodiment, a chemotherapeutic agent isan agent of use in treating neoplasms such as solid tumors. In oneembodiment, a chemotherapeutic agent is radioactive molecule. One ofskill in the art can readily identify a chemotherapeutic agent of use(e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 inHarrison's Principles of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) ed., © 2000Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology PocketGuide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; FischerD S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook,4th ed. St. Louis, Mosby-Year Book, 1993).

Cytokine. Proteins made by cells that affect the behavior of othercells, such as lymphocytes. In one embodiment, a cytokine is achemokine, a molecule that affects cellular trafficking.

Dendritic cell (DC): Dendritic cells are the principle antigenpresenting cells (APCs) involved in primary immune responses. Theirmajor function is to obtain antigen in tissues, migrate to lymphoidorgans and present the antigen in order to activate T cells.

When an appropriate maturational cue is received, DC are signaled toundergo rapid morphological and physiological changes that facilitatethe initiation and development of immune responses. Among these are theup-regulation of molecules involved in antigen presentation; productionof pro-inflammatory cytokines, including IL-12, key to the generation ofTh1 responses; and secretion of chemokines that help to drivedifferentiation, expansion, and migration of surrounding naive Th cells.Collectively, these up-regulated molecules facilitate the ability of DCto coordinate the activation and effector function of other surroundinglymphocytes that ultimately provide protection for the host. Althoughthe process of DC maturation is commonly associated with events thatlead to the generation of adaptive immunity, many stimuli derived fromthe innate branch of the immune system are also capable of activating DCto initiate this process. In this manner, DC provide a link between thetwo branches of the immune response, in which their initial activationduring the innate response can influence both the nature and magnitudeof the ensuing adaptive response. A dendritic cell precursor is a cellthat matures into an antigen presenting dendritic cell. In oneembodiment, a dendritic cell is a plasmacytoid dendritic cell.

Differentiation: The process by which cells become more specialized toperform biological functions, and differentiation is a property that istotally or partially lost by cells that have undergone malignanttransformation. For example, dendritic cell precursors undergomaturation to become APCs.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody binds a particular antigenicepitope.

Functionally Equivalent: Sequence alterations, for example in a D typeODN, that yield the same results as described herein. Such sequencealterations can include, but are not limited to, deletions, basemodifications, mutations, labeling, and insertions.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell to a stimulus. In one embodiment, the response is specificfor a particular antigen (an “antigen-specific response”).

Immune system deficiency: A disease or disorder in which the subject'simmune system is not functioning in normal capacity or in which it wouldbe useful to boost a subject's immune response. Immune systemdeficiencies include those diseases or disorders in which the immunesystem is not functioning at normal capacity, or in which it would beuseful to boost the immune system response. In one specific,non-limiting example, a subject with an immune system deficiency has atumor or cancer (e.g. tumors of the brain, lung (e.g. small cell andnon-small cell), ovary, breast, prostate, colon, as well as othercarcinomas and sarcomas).

Infectious agent: An agent that can infect a subject, including, but notlimited to, viruses, bacteria, and fungi.

Examples of infectious virus include: Retroviridae; Picornaviridae (forexample, polio viruses, hepatitis A virus; enteroviruses, humancoxsackie viruses, rhinoviruses, echoviruses); Calciviridae (such asstrains that cause gastroenteritis); Togaviridae (for example, equineencephalitis viruses, rubella viruses); Flaviridae (for example, dengueviruses, encephalitis viruses, yellow fever viruses); Coronaviridae (forexample, coronaviruses); Rhabdoviridae (for example, vesicularstomatitis viruses, rabies viruses); Filoviridae (for example, ebolaviruses); Paramyxoviridae (for example, parainfluenza viruses, mumpsvirus, measles virus, respiratory syncytial virus); Orthomyxoviridae(for example, influenza viruses); Bungaviridae (for example, Hantaanviruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae(hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviursesand rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpessimplex virus (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovirus(CMV), herpes viruses); Poxyiridae (variola viruses, vaccinia viruses,pox viruses); and Iridoviridae (such as African swine fever virus); andunclassified viruses (for example, the etiological agents of Spongiformencephalopathies, the agent of delta hepatitis (thought to be adefective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses).

Examples of infectious bacteria include: Helicobacter pyloris, Boreliaburgdorferi, Legionella pneumophilia, Mycobacteria sps (such as. M.tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusantracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, andActinomyces israelli.

Examples of infectious fungi include, but are not limited to,Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.

Other infectious organisms (such as protists) include: Plasmodiumfalciparum and Toxoplasma gondii.

Interferon alpha: At least 23 different variants of IFN-α are known. Theindividual proteins have molecular masses between 19-26 kDa and consistof proteins with lengths of 156-166 and 172 amino acids. All IFN-αsubtypes possess a common conserved sequence region between amino acidpositions 115-151 while the amino-terminal ends are variable. Many IFN-αsubtypes differ in their sequences at only one or two positions.Naturally occurring variants also include proteins truncated by 10 aminoacids at the carboxy-terminal end.

There are at least 23 different IFN-α genes. They have a length of 1-2kb and are clustered on human chromosome 9p22. Based upon the structurestwo types of IFN-alpha genes, designated class I and II, aredistinguished. They encode proteins of 156-166 and 172 amino acids,respectively.

IFN-α is assayed by a cytopathic effect reduction test employing humanand bovine cell lines. Minute amounts of IFN-α can be assayed also bydetection of the Mx protein specifically induced by this interferon. Asandwich ELISA employing bi-specific monoclonal antibodies for rapiddetection is also available.

Interferon gamma: IFN-γ is a dimeric protein with subunits of 146 aminoacids. The protein is glycosylated at two sites, and the pI is 8.3-8.5.IFN-γ is synthesized as a precursor protein of 166 amino acids includinga secretory signal sequence of 23 amino acids. Two molecular forms ofthe biologically active protein of 20 and 25 kDa have been described.Both of them are glycosylated at position 25. The 25 kDa form is alsoglycosylated at position 97. The observed differences of natural IFN-γwith respect to molecular mass and charge are due to variableglycosylation patterns. 40-60 kDa forms observed under non-denaturingconditions are dimers and tetramers of IFN-γ. The human gene has alength of approximately 6 kb. It contains four exons and maps tochromosome 12q24.1.

IFN-γ can be detected by sensitive immunoassays, such as an ELISA testthat allows detection of individual cells producing IFN-γ. Minuteamounts of IFN-γ can be detected indirectly by measuring IFN-inducedproteins such as Mx protein. The induction of the synthesis of IP-10 hasbeen used also to measure IFN-gamma concentrations. In addition,bioassays can be used to detect IFN-γ, such as an assay that employsinduction of indoleamine 2,3-dioxygenase activity in 2D9 cells.

Interferon Inducible Protein 10: A cytokine that is 98 amino acids inlength that has homology to platelet factor-4, and is a chemokine. Thehuman IP-10 genes contains four exons and maps to chromosome 4q12-21.

Interleukin-10: IL-10 is a homodimeric protein with subunits having alength of 160 amino acids that is a cytokine. Human IL-10 shows 73percent amino acid homology with murine IL-10. The human IL-10 genecontains four exons.

IL10 inhibits the synthesis of a number of cytokines such as IL-2 andIFN-γ in Th1 subpopulations of T-cells but not of Th2. IL10 can bedetected with an ELISA assay. In addition, the murine mast cell line D36can be used to bioassay human IL10. The intracellular factor can bedetected also by flow cytometry.

Isolated: An “isolated” biological component (such as a nucleic acid,peptide or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins which have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Leukocyte: Cells in the blood, also termed “white cells,” that areinvolved in defending the body against infective organisms and foreignsubstances. Leukocytes are produced in the bone marrow. There are 5 maintypes of white blood cell, subdivided between 2 main groups:polymorphomnuclear leukocytes (neutrophils, eosinophils, basophils) andmononuclear leukocytes (monocytes and lymphocytes). When an infection ispresent, the production of leukocytes increases.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Maturation: The process in which an immature cell, such as dendriticcell, changes in form or function to become a functional mature cell,such as an APC.

Neoplasm: An abnormal cellular proliferation, which includes benign andmalignant tumors, as well as other proliferative disorders.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle or double stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.

Oligonucleotide or “oligo”: Multiple nucleotides (i.e. moleculescomprising a sugar (e.g. ribose or deoxyribose) linked to a phosphategroup and to an exchangeable organic base, which is either a substitutedpyrimidine (Py) (e.g. cytosine (C), thymine (T) or uracil (U)) or asubstituted purine (Pu) (e.g. adenine (A) or guanine (G)). The term“oligonucleotide” as used herein refers to both oligoribonucleotides(ORNs) and oligodeoxyribonucleotides (ODNs). The term “oligonucleotide”also includes oligonucleosides (i.e. an oligonucleotide minus thephosphate) and any other organic base polymer. Oligonucleotides can beobtained from existing nucleic acid sources (e.g. genomic or cDNA), butare preferably synthetic (e.g. produced by oligonucleotide synthesis).

A “stabilized oligonucleotide” is an oligonucleotide that is relativelyresistant to in vivo degradation (for example via an exo- orendo-nuclease). In one embodiment, a stabilized oligonucleotide has amodified phosphate backbone. One specific, non-limiting example of astabilized oligonucleotide has a phosphorothioate modified phosphatebackbone (wherein at least one of the phosphate oxygens is replaced bysulfur). Other stabilized oligonucleotides include: nonionic DNAanalogs, such as alkyl- and aryl-phosphonates (in which the chargedphosphonate oxygen is replaced by an alkyl or aryl group), phophodiesterand alkylphosphotriesters, in which the charged oxygen moiety isalkylated. Oligonucleotides which contain a diol, such astetraethyleneglycol or hexaethyleneglycol, at either or both terminihave also been shown to be substantially resistant to nucleasedegradation.

An “immunostimulatory oligonucleotide,” “immunostimulatory CpGcontaining oligodeoxynucleotide,” “CpG ODN,” refers to anoligodeoxynucleotide, which contains a cytosine, guanine dinucleotidesequence and stimulates (e.g. has a mitogenic effect or induces cytokineproduction) vertebrate immune cells. The cytosine, guanine isunmethylated.

An “oligonucleotide delivery complex” is an oligonucleotide associatedwith (e.g. ionically or covalently bound to; or encapsulated within) atargeting means (e.g. a molecule that results in a higher affinitybinding to a target cell (e.g. B-cell or natural killer (NK) cell)surface and/or increased cellular uptake by target cells). Examples ofoligonucleotide delivery complexes include oligonucleotides associatedwith: a sterol (e.g. cholesterol), a lipid (e.g. cationic lipid,virosome or liposome), or a target cell specific binding agent (e.g. aligand recognized by a target cell specific receptor). Preferredcomplexes must be sufficiently stable in vivo to prevent significantuncoupling prior to internalization by the target cell. However, thecomplex should be cleavable or otherwise accessible under appropriateconditions within the cell so that the oligonucleotide is functional.(Gursel, J. Immunol. 167: 3324, 2001)

Pharmaceutical agent or drug: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject. Pharmaceutical agents include, but are notlimited to, chemotherapeutic agents and anti-infective agents.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this invention are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the fusion proteins hereindisclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Preventing or treating a disease: “Preventing” a disease refers toinhibiting the full development of a disease, for example in a personwho is known to have a predisposition to a disease such as an autoimmunedisorder. An example of a person with a known predisposition is someonewith a history of diabetes in the family, or who has been exposed tofactors that predispose the subject to a condition, such as lupus orrheumatoid arthritis. “Treatment” refers to a therapeutic interventionthat ameliorates a sign or symptom of a disease or pathologicalcondition after it has begun to develop.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell.Preferably, a preparation is purified such that the protein or peptiderepresents at least 50% of the total peptide or protein content of thepreparation.

Self-complementary nucleic acid sequence: A nucleic acid sequence thatcan form Watson-Crick base pairs. The four bases characteristic ofdeoxyribonucleic unit of DNA are the purines (adenine and guanine) andthe pyrimidines (cytosine and thymine). Adenine pairs with thymine viatwo hydrogen bonds, while guanine pairs with cytosine via three hydrogenbonds. If a nucleic acid sequence includes two or more bases in sequencethat can form hydrogen bonds with two or more other bases in the samenucleic acid sequence, then the nucleic acid includes aself-complementary sequence. In several embodiments, aself-complementary nucleic acid sequence includes 3, 4, 5, 6 or morebases that could form hydrogen bonds with 3, 4, 5, 6 or more bases,respectively, of the same nucleic acid sequence.

Therapeutically effective dose: A dose sufficient to preventadvancement, or to cause regression of the disease, or which is capableof relieving symptoms caused by the disease, such as pain or swelling.

Vaccine: A preparation of attenuated microorganisms (including but notlimited to bacteria and viruses), living microorganisms, antigen, orkilled microorganisms, administered for the prevention, amelioration ortreatment of infectious disease.

A. CpG Oligodeoxynucleotides

K type CpG ODNs have been previously described. K ODNs which exhibit thegreatest immunostimulatory activity share specific characteristics.These characteristics differ from those of the Formula II or D ODN (seebelow). In addition, K ODN have specific effects on the cells of theimmune system, which differ from the effects of D ODN. For example, KODN stimulate proliferation of B cells and stimulate the production ofIL-6.

The K ODNs at least about 10 nucleotides and include a sequencerepresented by either Formula I:

5′N₁N₂N₃T-CpG-WN₄N₅N₆3′

wherein the central CpG motif is unmethylated, W is A or T, and N₁, N₂,N₃, N₄, N₅, and N₆ are any nucleotides.

These Formula I or K ODN, stimulate B cell proliferation and thesecretion of IgM and IL-6, processes involved in the body's humoralimmunity, such as the production of antibodies against foreign antigens.In one embodiment, the K ODNs induce a humoral immune response.

In one embodiment, K type oligonucleotides of the formula

5′N₁N₂N₃T-CpG-WN₄N₅N₆3′

contain a phosphate backbone modification. In one specific, non-limitingexample, the phosphate backbone modification is a phosphorothioatebackbone modification (i.e., one of the non-bridging oxygens is replacedwith sulfur, as set forth in International Patent Application WO95/26204, herein incorporated by reference). In one embodiment, K ODNshave a phosphorothioate backbone, and at least one unmethylated CpGdinucleotide. Eliminating the CpG dinucleotide motif from the K ODNsignificantly reduces immune activation. Incorporating multiple CpGs ina single K ODN increases immune stimulation. Preferably, the K ODN areat least 12 bases long. In addition, K ODN containing CpG motifs at the5′ end are the most stimulatory, although at least one base upstream ofthe CpG is required. More particularly, the most active K ODNs contain athymidine immediately 5′ from the CpG dinucleotide, and a TpT or a TpAin a position 3′ from the CpG motif. Modifications which are greaterthan 2 base pairs from the CpG dinucleotide motif appear to have littleeffect on K ODN activity.

D type ODNs differ both in structure and activity from K type ODNs. Theunique activities of D type ODNs are disclosed below (see section C).For example, as disclosed herein, D oligodeoxynucleotides stimulate therelease of cytokines from cells of the immune system. In specific,non-limiting examples D type oligonucleotides stimulate the release orproduction of IP-10 and IFN-α by monocytes and/or plasmacitoid dendriticcells and the release or production of IFN-γ by NK cells. Thestimulation of NK cells by D oligodeoxynucleotides can be either director indirect.

With regard to structure, in one embodiment, a CpG motif in a D typeoligonucleotides has been described by Formula II:

5′RY-CpG-RY3′

wherein the central CpG motif is unmethylated, R is A or G (a purine),and Y is C or T (a pyrimidine). D-type oligonucleotides include anunmethylated CpG dinucleotide. Inversion, replacement or methylation ofthe CpG reduces or abrogates the activity of the D oligonucleotide.

In one embodiment, a D type ODN is at least about 16 nucleotides inlength and includes a sequence represented by Formula III:

5′X₁X₂X₃Pu₁Py₂CpGPu₃Py₄X₄X₅X₆(W)_(M)(G)_(N)-3′

wherein the central CpG motif is unmethylated, Pu is a purinenucleotide, Py is a pyrimidine nucleotide, X and W are any nucleotide, Mis any integer from 0 to 10, and N is any integer from 4 to 10.

The region Pu₁ Py₂ CpG Pu₃ Py₄ is termed the CpG motif. The regionX₁X₂X₃ is termed the 5′ flanking region, and the region X₄X₅X₆ is termedthe 3′ flanking region. If nucleotides are included 5′ of X₁X₂X₃ in theD ODN these nucleotides are termed the 5′ far flanking region.Nucleotides 3′ of X₄X₅X₆ in the D ODN are termed the 3′ far flankingregion.

In one specific non-limiting example, Py₂ is a cytosine. In anotherspecific, non-limiting example, Pu₃ is a guanidine. In yet anotherspecific, non limiting example, Py₂ is a thymidine and Pu₃ is anadenine. In a further specific, non-limiting example, Pu₁ is an adenineand Py₂ is a tyrosine. In another specific, non-limiting example, Pu₃ isan adenine and Py₄ is a tyrosine.

In one specific not limiting example, N is from about 4 to about 8. Inanother specific, non-limiting example, N is about 6.

D-type CpG oligonucleotides can include modified nucleotides. Withoutbeing bound by theory, modified nucleotides can be included to increasethe stability of a D-type oligonucleotide. Without being bound bytheory, because phosphorothioate-modified nucleotides confer resistanceto exonuclease digestion, the D ODN are “stabilized” by incorporatingphosphorothioate-modified nucleotides. In one embodiment, the CpGdinucleotide motif and its immediate flanking regions includephosphodiester rather than phosphorothioate nucleotides. In one specificnon-limiting example, the sequence Pu₁ Py₂ CpG Pu₃ Py₄ includesphosphodiester bases. In another specific, non-limiting example, all ofthe bases in the sequence Pu₁ Py₂ CpG Pu₃ Py₄ are phosphodiester bases.In yet another specific, non-limiting example, X₁X₂X₃ and X₄X₅X₆(W)_(m)(G)_(N) include phosphodiester bases. In yet another specific,non-limiting example, X₁X₂X₃ Pu₁ Py₂ CpG Pu₃ Py₄X₄X₅X₆(W)_(M) (G)_(N)include phosphodiester bases. In further non-limiting examples thesequence X₁X₂X₃ includes at most one or at most two phosphothioate basesand/or the sequence X₄X₅X₆ includes at most one or at most twophosphothioate bases. In additional non-limiting examples, X₄X₅X₆(W)_(M)(G)_(N) includes at least 1, at least 2, at least 3, at least 4, or atleast 5 phosphothioate bases. Thus, a D type oligodeoxynucleotide can bea phosphorothioate/phosphodiester chimera.

As disclosed herein, any suitable modification can be used in thepresent invention to render the D oligodeoxynucleotide resistant todegradation in vivo (e.g., via an exo- or endo-nuclease). In onespecific, non-limiting example, a modification that renders theoligodeoxynucleotide less susceptible to degradation is the inclusion ofnontraditional bases such as inosine and quesine, as well as acetyl-,thio- and similarly modified forms of adenine, cytidine, guanine,thymine, and uridine. Other modified nucleotides include nonionic DNAanalogs, such as alkyl or aryl phosphonates (i.e., the chargedphosphonate oxygen is replaced with an alkyl or aryl group, as set forthin U.S. Pat. No. 4,469,863), phosphodiesters and alkylphosphotriesters(i.e., the charged oxygen moiety is alkylated, as set forth in U.S. Pat.No. 5,023,243 and European Patent No. 0 092 574). Oligonucleotidescontaining a diol, such as tetraethyleneglycol or hexaethyleneglycol, ateither or both termini, have also been shown to be more resistant todegradation. The D type oligodeoxynucleotides can also be modified tocontain a secondary structure (e.g., stem loop structure). Without beingbound by theory, it is believed that incorporation of a stem loopstructure renders and oligodeoxynucleotide more effective.

In a further embodiment, Pu₁ Py₂ and Pu₃ Py₄ are self-complementary. Inanother embodiment, X₁X₂X₃ and X₄X₅X₆ are self complementary. In yetanother embodiment X₁X₂X₃Pu₁ Py₂ and Pu₃ Py₄ X₄X₅X₆ are selfcomplementary.

Specific non-limiting examples of a D type oligonucleotide wherein Pu₁Py₂ and Pu₃ Py₄ are self-complementary include, but are not limited to,ATCGAT, ACCGGT, ATCGAC, ACCGAT, GTCGAC, or GCCGGC. Without being boundby theory, the self-complementary base sequences can help to form astem-loop structure with the CpG dinucleotide at the apex to facilitateimmunostimulatory functions. Thus, in one specific, non-limitingexample, D type oligonucleotides wherein Pu₁ Py₂ and Pu₃ Py₄ areself-complementary induce higher levels of IFN-γ production from a cellof the immune system (see below). The self-complementary need not belimited to Pu₁ Py₂ and Pu₃ Py₄. Thus, in another embodiment, additionalbases on each side of the three bases on each side of the CpG-containinghexamer form a self-complementary sequence (see above).

One specific, non-limiting example of a sequence wherein Pu₁ Py₂ and Pu₃Py₄ are self-complementary but wherein the far-flanking sequences arenot self-complementary is

(ODN D 113, SEQ ID NO: 11) GGTGCATCGATACAGGGGGG.

This oligodeoxynucleotide has a far flanking region that is not selfcomplementary and induces high levels of IFN-γ and IFN-α.

Another specific, non-limiting example of a D oligodeoxynucleotides is:

GGTGCGTCGATGCAGGGGGG. (D28, SEQ ID NO: 7)

This oligodeoxynucleotide is of use for inducing production and/orrelease of cytokines from immune cells, although it lacks aself-complementary motif.

In one embodiment, the D type oligodeoxynucleotides disclosed herein areat least about 16 nucleotides in length. In a second embodiment, a Dtype oligodeoxynucleotide is at least about 18 nucleotides in length. Inanother embodiment, a D type oligodeoxynucleotide is from about 16nucleotides in length to about 100 nucleotides in length. In yet anotherembodiment, a D type oligodeoxynucleotide is from about 16 nucleotidesin length to about 50 nucleotides in length. In a further embodiment, aD type oligodeoxynucleotide is from about 18 nucleotides in length toabout 30 nucleotides in length.

In another embodiment, the oligodeoxynucleotide is at least 18nucleotides in length, and at least two G's are included at the 5′ endof the molecule, such that the oligodeoxynucleotide includes a sequencerepresented by Formula IV:

5′GGX₁X₂X₃Pu₁Py₂CpGPu₃Py₄X₄X₅X₆(W)_(M)(G)_(N)-3′.

The D type oligodeoxynucleotide can include additional G's at the 5′ endof the oligodeoxynucleotide. In one specific example, about 1 or about 2G's are included at the 5′ end of an oligodeoxynucleotide including asequence as set forth as Formula IV.

Examples of a D type oligodeoxynucleotide include, but are not limitedto,

5′XXTGCATCGATGCAGGGGGG 3′ (SEQ ID NO: 12) 5′XXTGCACCGGTGCAGGGGGG3′,(SEQ ID NO: 13) 5′XXTGCGTCGACGCAGGGGGG3′, (SEQ ID NO: 14)5′XXTGCGTCGATGCAGGGGGG3′, (SEQ ID NO: 16) 5′XXTGCGCCGGCGCAGGGGGG3′,(SEQ ID NO: 17) 5′XXTGCGCCGATGCAGGGGGG3′, (SEQ ID NO: 18)5′XXTGCATCGACGCAGGGGGG3′, (SEQ ID NO: 19) 5′XXTGCGTCGGTGCAGGGGGG3′,(SEQ ID NO: 20)wherein X any base, or is no base at all. In one specific, non-limitingexample, X is a G.

The oligodeoxynucleotides disclosed herein can be synthesized de novousing any of a number of procedures well known in the art. For example,the oligodeoxynucleotides can be synthesized as set forth in U.S. Pat.No. 6,194,388, which is herein incorporated by reference in itsentirety. A D type oligodeoxynucleotide may be synthesized using, forexample, the B-cyanoethyl phosphoramidite method or nucleosideH-phosphonate method. These chemistries can be performed by a variety ofautomated oligonucleotide synthesizers available in the market.Alternatively, oligodeoxynucleotides can be prepared from existingnucleic acid sequences (e.g. genomic or cDNA) using known techniques,such as employing restriction enzymes, exonucleases or endonucleases,although this method is less efficient than direct synthesis.

B. Delivery Complexes and Pharmaceutical Compositions

In one embodiment, a D type oligodeoxynucleotide is included in deliverycomplex. The delivery complex can include the D type ODN and a targetingmeans. Any suitable targeting means can be used. For example, a D typeoligodeoxynucleotide can be associated with (e.g., ionically orcovalently bound to, or encapsulated within) a targeting means (e.g., amolecule that results in higher affinity binding to a target cell, suchas a B cell). A variety of coupling or cross-linking agents can be usedto form the delivery complex, such as protein A, carbodiamide, andN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). Examples of anoligodeoxynucleotide delivery complexes include a D typeoligodeoxynucleotide associated with a sterol (e.g., cholesterol), alipid (e.g., a cationic lipid, anionic lipid, virosome or liposome), anda target cell specific binding agent (e.g., a ligand recognized bytarget cell specific receptor). Without being bound by theory, thecomplex is sufficiently stable in vivo to prevent significant uncouplingprior to delivery to the target cell. In one embodiment, the deliverycomplex is cleavable such that the oligodeoxynucleotide is released in afunctional form at the target cells.

In one embodiment, a pharmacological composition is provided thatincludes a D type oligonucleotide and a pharmacologically acceptablecarrier. Pharmacologically acceptable carriers (e.g., physiologically orpharmaceutically acceptable carriers) are well known in the art. Asuitable pharmacological composition can be formulated to facilitate theuse of a D type ODN in vivo and/or ex vivo. Such a composition can besuitable for delivery of the active ingredient to any suitable host,such as a patient for medical application, and can be manufactured in amanner that is itself known, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmacological compositions for use can be formulated in a conventionalmanner using one or more pharmacologically (e.g., physiologically orpharmaceutically) acceptable carriers comprising excipients, as well asoptional auxiliaries that facilitate processing of the active compoundsinto preparations which can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen, and whether usewill be an in vivo or an ex vivo use. For use in vivo, administrationcan be either systemic or local. In addition, one of skill in the artcan readily select a suitable route of administration, including, butnot limited to intravenous, intramuscular, intraperitioneal,transmucosal, subcutaneous, transdermal, transnasal, and oraladministration.

Thus, for injection, the active ingredient can be formulated in aqueoussolutions, preferably in physiologically compatible buffers. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art. For oral administration, the active ingredient can becombined with carriers suitable for inclusion into tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike. For administration by inhalation, the active ingredient isconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant.The active ingredient can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Similarly, Dtype oligodeoxynucleotides can be formulated for intratracheal or forinhalation. Such compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Other pharmacological excipients are known in the art.

C. Method of Inducing an Immune Response

A method is disclosed herein for stimulating a cell of the immunesystem. The method includes contacting the cell with an effective amountof a D type oligodeoxynucleotide disclosed herein, thereby stimulatingthe cell (see also PCT Application Nos. WO0061151A3, WO9956755A1,WO9840100A1, WO9818810A1, WO0122990A2; which are herein incorporated byreference in their entirety).

In contrast, D type oligodeoxynucleotides have differentimmunostimulatory activities. As disclosed herein, administration of a Dtype oligodeoxynucleotide activates monocytes and/or natural killercells, and induces the maturation of dendritic cells. Furthermore, a Dtype oligodeoxynucleotide can be used to increase the production ofcytokines (for example IP-10, IFN-α or IFN-γ) by a cell of the immunesystem.

Administration of the D type oligodeoxynucleotide can be by any suitablemethod. For example, the ODN can be administered in vivo or ex vivo.

Thus, in one embodiment, a method is also disclosed herein for producingan immune response in a subject. The subject can be any mammal,particularly a primate, such as a human. The method includesadministering a D type oligodeoxynucleotide to the subject, therebyinducing the immune response. In yet one embodiment, the immune responseincludes induction of the maturation of a dendritic cell or theactivation of a natural killer cell and/or a monocyte. In a furtherembodiment, the immune response includes the production of a cytokine,such as, for example, IL-10, IP-10, IFN-α or IFN-γ.

In one embodiment, a method is provided for inducing an immune responsein a subject wherein the method includes contacting a monocyte or adendritic cell precursor in vitro with a D type oligodeoxynucleotide toproduce an activated antigen presenting cell. The monocytes or dendriticcell precursors can be contacted with the D type oligodeoxynucleotidesin the presence of or in the absence of antigen. The activated antigenpresenting cell is then administered to the subject to induce an immuneresponse.

In another embodiment, a method is provided herein for inducing animmune response in a subject that includes contacting a monocyte or adendritic cell precursor in vitro with a D type oligodeoxynucleotide toproduce an activated antigen presenting cell. The monocytes or dendriticcell precursors can be contacted with the D type oligodeoxynucleotidesin the presence of or in the absence of antigen. Lymphocytes or naturalkiller are then contacted with the activated antigen presenting cells invitro, or with cytokines secreted by the activated antigen presentingcells in vitro, to produce activated lymphocytes or activated naturalkiller cells. The activated lymphocytes or natural killer cells areadministered to the subject to induce the immune response.

In order to induce an immune response, a D type oligodeoxynucleotide isadministered either alone or in conjunction with another molecule.Co-administration includes administering the molecule and the D typeoligodeoxynucleotide at the same time, or sequentially. The othermolecule can be any other agent, such as a protein, an antigenicepitope, a hydrocarbon, lipid, mitogen, an anti-infectious agent (suchas antiviral, antifungal, or anti-bacterial agent) or a vaccine (such asa live, attenuated, or heat-killed vaccine).

Without being bound by theory, when administered to a subject, it isbelieved that the ODNs initially act on antigen presenting cells (e.g.,B cells, macrophages and dendritic cells). These cells then releasecytokines, which activate natural killer (NK) cells. The activation ofnatural killer cells may be direct (e.g. through contact of the NK cellwith a D type ODN) or indirect (e.g. by activating the secretion ofcytokines, which then activate the natural killer cells). Either acell-mediated or humoral immune response then occurs in the host.

As disclosed herein, D type oligodeoxynucleotides are a unique type ofCpG containing oligodeoxynucleotides that have specific effects on thecells of the immune system. For several examples, D typeoligonucleotides activate natural killer cells and monocytes, and inducethe maturation of dendritic cells. In other examples, D typeoligodeoxynucleotides increase production of cytokines such as IP-10,IL-10, IFNγ and IFN-γ. These effects are different from the effects of Ktype oligodeoxynucleotides. As previously described, K typeoligodeoxynucleotides support B cell proliferation, and induce theproduction of IL-6. Thus, the elucidation of the immunostimulatoryeffects of D type oligodeoxynucleotides allows for customizing ortailoring of the type of immune obtained by administration ofoligodeoxynucleotides. For example, a K type oligodeoxynucleotide can beutilized when production of IL-6 is desired, whereas D typeoligodeoxynucleotides can be used when production of IFN-γ is desired.

In one embodiment, a D type oligodeoxynucleotide is administered to asubject, such as a subject that has an autoimmune disease. Specific,non-limiting examples of autoimmune diseases include, but are notlimited to diabetes, rheumatoid arthritis, lupus erythematosus, andmultiple sclerosis. In one embodiment, the subject has cancer.

Also disclosed herein are methods of use to treat, prevent, orameliorate an allergic reaction in a subject. An allergy refers to anacquired hypersensitivity to a substance (i.e., an allergen). Allergicconditions include eczema, allergic rhinitis or coryza, hay fever,bronchial asthma, uticaria (hives), food allergies, and other atopicconditions. The list of allergens is extensive and includes pollens,insect venoms, animal dander, dust, fungal spores, and drugs (e.g.,penicillin). Examples of natural, animal, and plant allergens can befound in International Patent Application WO 98/18810. In one embodimenta D-type oligodeoxynucleotide administered to a subject to treat anallergic condition such as allergic asthma. In another embodiment,

the D type oligodeoxynucleotide is administered in combination with anysuitable anti-allergenic agent. Suitable anti-allergenic agents includethose substances given in treatment of the various allergic conditionsdescribed above, examples of which can be found in the Physicians' DeskReference (1998).

In another embodiment, a D type oligodeoxynucleotide is administered toa subject that has a neoplasm. The D-type oligodeoxynucleotide isadministered either alone or in combination with any suitableanti-neoplastic agent, such as a chemotherapeutic agent or radiation.Suitable neoplasms include, but are not limited to, solid tumors such ascancers of the brain, lung (e.g., small cell and non-small cell), ovary,breast, prostate, and colon, as well as carcinomas and sarcomas. Withoutbeing bound by theory, it is believed that the D typeoligodeoxynucleotide increases the immune response to the neoplasm, andthus is involved in the reduction of tumor burden.

In a further embodiment, a method is provided to enhance the efficacy ofany suitable vaccine. Suitable vaccines include those directed againstLeishmania, Hepatitis A, B, and C, examples of which can be found in thePhysicians' Desk Reference (1998), and DNA vaccines directed against,for example, malaria. (See generally Klinman et al., 17 Vaccine 17: 19,1999; McCluskie and Davis, J. Immun. 161:4463, 1998).

D type oligodeoxynucleotides can be used to treat, prevent, orameliorate any condition associated with an infectious agent. The D typeoligodeoxynucleotide can be administered to a subject infected with theinfectious agent alone or in combination with any suitableanti-infectious agent, such as an antiviral, anti-fungal oranti-bacterial agent (see Physicians' Desk Reference, 1998). Specific,non-limiting examples of infectious agents conditions associated withinfectious agents are tularemia, francisella, schistosomiasis,tuberculosis, malaria, and leishmaniasis. Examples of infectious agentsare viruses, bacteria, fungi, and other organisms (e.g., protists) canbe found in International Patent Application WO 98/18810.

The D type oligodeoxynucleotides disclosed herein can also be used withany suitable antisense therapy. Suitable antisense agents are those thatspecifically bind either with a target DNA or a target RNA and inhibitexpression of the target sequence (see Lonnberg et al., Ann. Med. 28:511, 1996; Alama et al., Pharmacol. Res. 36: 171, 1997; Scanlon et al.,FASEB J. 9: 1288, 1995; Oberbauer, 109 Wien Klin Wochenschr 109: 40,1997).

The present invention is further described in the following examples.These examples are intended only to illustrate the invention and are notintended to limit the scope of the invention in any way.

EXAMPLES Example 1 Cytokine Production

The oligodeoxynucleotides disclosed herein can be used to produce animmune response. In one embodiment, an immune response can be measuredby cytokine production. As shown herein, K type oligodeoxynucleotidescan be used to increase the production of the cytokines IL-6 and inducecell proliferation. D type oligodeoxynucleotides induce the productionof IFN-γ.

Human peripheral blood mononuclear cells (PBMC) were isolated, asdescribed elsewhere (Ballas et al., J. Allergy Clin. Immunol. 85: 453,1990; Ballas a Rasmussen, J. Immunol. 45:1039, 1990; Ballas andRasmussen, J. Immunol. 150: 17, 1993). Oligodeoxynucleotides (ODNs) weresynthesized on a DNA synthesizer (Applied Biosystems Inc., Foster City,Calif.), as described elsewhere (Beacage and Caruthers, TetrahedronLetters 22: 1859, 1981). In some ODNs, the normal DNA backbonephosphodiesterase linkages were replaced with phosphorothioate linkages,as described elsewhere (Agrawal et al., Proc. Natl. Acad. Sci. USA 94:2620, 1997; Agrawal TIB TECH 14: 376, 1996). To reduce degradation ofthe ODNs, those that did not have an entire phosphorothioate backbonecontained phosphorothioate linkages at the 5′ and 3′ ends. Cells wereincubated for approximately 72 hrs with the various ODNs. IL-6 and TNF-γlevels were determined by ELISA using anti-IL-6 and anti-TNF-γantibodies, as described elsewhere (Maniatis et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York,1989). Cell proliferation was determined by [³H] thymidineincorporation, as described elsewhere (Liang et al., J. Clin. Invest.98:1121, 1996).

IL-6 levels and cell proliferation are set forth in Table 1: Inductionof a Humoral Immune Response In Vitro. These data demonstrate that asequence containing 5′ N₁N₂N₃T-CpG-WN₄N₅N₆ 3′, wherein the central CpGmotif is unmethylated, W is A or T, and N₁, N₂, N₃, N₄, N₅, and N₆ areany nucleotides, (K type oligodeoxynucleotides) induce a the productionof IL-6 and a humoral immune response. However, D typeoligodeoxynucleotides induce only low levels of IL-6 production.

In addition, maximum induction was observed for K type ODNs thatcontained a phosphorothioate backbone. IFN-γ levels and cellproliferation are set forth in Table 1. These data demonstrate that Dtype oligonucleotides increase production of IFN-γ. Maximum inductionoccurred with ODNs containing phosphodiesterase linkages.

TABLE 1Underlined bases are phosphodiester. * indicates methylated CG. Boldindicates self-complementary sequences. Sequence identifier is noted below thenucleic acid sequence. For each heading (IFN-g, IL-6, IgM, PROLIF (prolifer-)ation) first column is average and second column is standard deviation.ODN SEQUENCE IFN-g* IL-6* IgM* PROLIF* media (None) 0.9 0.8 0.7 1 0.50.5 0.9 0.6 D ODN DV104 GGTGCATCGATGCAGGGGGG 47.7 64.7 30.8 31 10.2 12.32.9 1.7 (SEQ ID NO: 1) DV19 GGTGCATCGATGCAGGGGGG 44.0 71.8 12.5 17 9.621.0 3.1 3.1 (SEQ ID NO: 1) DV29 GGTGCACCGGTGCAGGGGGG 35.9 38.9 3.8 42.5 2.9 1.7 1.2 (SEQ ID NO: 2) DV35 GGTGCATCGATGCAGGGGGG 33.3 63.6 28.029 5.7 8.4 2.0 1.1 (SEQ ID NO: 1) DV28 GGTGCGTCGATGCAGGGGGG 32.2 45.313.8 23 9.0 14.3 2.8 2.0 (SEQ ID NO: 7) DV106 GGTGTGTCGATGCAGGGGGG 29.971.4 7.5 6 32.2 94.5 2.9 1.8 (SEQ ID NO: 26) DV116 TGCATCGATGCAGGGGGG25.1 28.9 28.3 25 6.2 3.3 2.6 1.6 (SEQ ID NO: 12) DV113GGTGCATCGATACAGGGGGG 21.1 15.3 144.8 255 4.0 4.2 4.7 5.6 (SEQ ID NO: 11)DV34 GGTGCATCGATGCAGGGGGG 16.2 19.1 3.6 6 2.4 2.6 3.2 4.5 (SEQ ID NO: 1)DV102 GGTGCATCGTTGCAGGGGGG 15.8 39.4 6.6 7 24.0 58.0 2.1 1.1(SEQ ID NO: 30) DV32 GGTGCGTCGACGCAGGGGGG 14.5 18.0 2.7 5 1.4 2.1 2.80.9 (SEQ ID NO: 31) DV117 GGTCGATCGATGCACGGGGG 13.4 14.6 25.8 23 19.622.8 6.2 2.7 (SEQ ID NO: 32) DV37 GGTGC ATCGAT GCAGGGGGG 13.0 19.6 13.321 38.0 33.0 4.8 3.6 (SEQ ID NO: 1) DV25 GGTGCATCGATGCAGGGGGG 12.2 7.92.0 3 2.1 2.7 0.9 0.7 (SEQ ID NO: 1) DV30 GGTGCATCGACGCAGGGGGG 11.3 8.12.4 3 6.4 6.9 2.4 1.2 (SEQ ID NO: 35) dv120 GGTGCATCGATAGGCGGGGG 8.711.5 1.0 1 3.5 1.1 1.9 1.0 (SEQ ID NO: 36) DV27 GGTGCACCGATGCAGGGGGG 8.57.3 2.9 4 3.3 4.9 1.9 1.5 (SEQ ID NO: 37) dv119 CCTGCATCGATGCAGGGGGG 7.59.1 2.0 1 1.6 0.4 1.0 0.3 (SEQ ID NO: 38) D142 GGTATATCGATATAGGGGGG 7.31.1 15.9 5 22.5 5.0 2.0 1.0 (SEQ ID NO: 39) d143 GGTGGAT CG ATCCAGGGGGG6.4 4.5 16.0 8 47.0 12.0 2.0 1.0 (SEQ ID NO: 40) D CONTROLS dv17GGTGCAACGTTGCAGGGGGG 2.3 1.5 5.0 4 8.0 2.0 4.0 2.0 (SEQ ID NO: 41) DV78GGTGCATCGATAGAGGGGGG 0.8 0.5 2.0 4 4.2 3.0 1.4 0.9 (SEQ ID NO: 42) DV96GGTGCATCGTAGCAGGGGGG 1.1 0.7 1.5 2 1.1 2.9 1.2 0.3 (SEQ ID NO: 43) DV95GGTGGTTCGATGCAGGGGGG 2.1 2.2 2.5 3 1.2 1.8 1.2 0.7 (SEQ ID NO: 44) DV93GGTGCATCGATGCAGGGGGG 0.9 1.3 11.9 26 2.7 3.5 1.5 0.6 (SEQ ID NO: 1) DV92GGTGCACCGGTGCAAAAAAA 0.4 0.7 2.0 3 0.8 1.2 1.4 0.8 (SEQ ID NO: 46) DV81GGTGCATCGATAGAGGGG 0.8 1.1 1.2 2 1.2 1.6 1.4 0.6 (SEQ ID NO: 47) DV77GGTGCATCGATGCAGGGGGG 1.0 1.6 9.5 13 82.1 181.9 6.5 12.2 (SEQ ID NO: 1)DV76 GGTGCATCGATGCAAAAAAA 1.6 1.5 1.3 2 1.2 1.5 1.7 0.4 (SEQ ID NO: 49)DV71 GGGGTCGACAGGG 1.4 2.2 1.8 2 0.7 0.5 1.2 1.0 (SEQ ID NO: 50) DV49GGTGCATAAATGCAGGGGGG 1.5 1.2 1.3 2 1.0 1.0 1.2 1.0 (SEQ ID NO: 51) DV48GGTGCATCAATGCAGGGGGG 1.0 0.5 1.2 2 1.0 1.0 1.2 1.0 (SEQ ID NO: 52) DV47GGTGCATTGATGCAGGGGGG 1.2 0.5 1.5 2 1.0 1.0 1.2 1.0 (SEQ ID NO: 53) DV45GGTGCATC*GATGCAGGGGGG 3.4 2.9 7.0 14 0.8 1.5 1.3 0.3 (SEQ ID NO: 54)DV26 GGTGCATGCATGCAGGGGGG 7.6 15.3 0.0 0 0.6 1.0 1.3 1.4 (SEQ ID NO: 55)DV20 GGTGCATGCATGCAGGGGGG 2.3 4.2 2.1 2 1.0 1.8 1.1 0.8 (SEQ ID NO: 55)DV122 GGTGC ATTGATGCAGGGGGG 0.9 1.5 1.3 1 2.0 0.4 0.7 0.3(SEQ ID NO: 53) DV114 GGTGCACTGGTGCAGGGGGG 1.8 2.0 1.8 2 1.2 1.2 1.0 0.6(SEQ ID NO: 58) DV111 GGTGTATCGATGCAAAAGGG 5.8 5.1 12.3 13 4.8 7.6 4.48.0 (SEQ ID NO: 59) DV108 GGTGCCCCGTTGCAGGGGGG 1.2 1.4 1.7 3 1.0 1.5 1.00.2 (SEQ ID NO: 60) DV107 GGTGCAACGGGGCAGGGGGG 2.5 3.3 7.7 15 0.4 0.61.1 0.4 (SEQ ID NO: 61) DV105 AATGCATCGATGCAAAAAAA 1.1 1.8 2.4 3 1.2 1.51.0 0.4 (SEQ ID NO: 62) DV103 GGTGCACCGTGGCAGGGGGG 9.4 16.8 8.7 12 5.48.4 1.7 1.9 (SEQ ID NO: 63) DV100 GGTGCATCGAAGCAGGGGGG 3.4 3.5 7.2 621.0 24.0 9.1 5.9 (SEQ ID NO: 64) d79 GGTGGATCGATGCAGGGGGG 0.6 0.5 1.0 123.0 2.0 (SEQ ID NO: 65) d145 GGTGCACGCGTGCAGGGGGG 4.8 0.8 2.5 4 16.83.0 (SEQ ID NO: 5) d144 GGTGCATGTATGCAGGGGGG 0.0 0.0 0.6 1 1.3 2.0(SEQ ID NO: 67) AA20 GGGGGATCGATGGGGG 2.7 5.1 3.1 3 2.2 2.5 1.6 1.2(SEQ ID NO: 68) AA3M GGGGGAAGCTTCGGGG 2.2 3.2 1.1 2 0.4 0.8 1.2 1.0(SEQ ID NO: 69) K ODN K22 CTCGAGCGTTCTC 9.3 1.8 43.3 20 131.8 94.0 18.33.3 (SEQ ID NO: 70) DV84 ACTCTCGAGCGTTCTA 1.1 1.6 42.6 91 79.7 105.318.5 25.8 (SEQ ID NO: 71) K21 TCTCGAGCGTTCTC 7.2 2.8 39.6 17 237.6 194.234.6 9.3 (SEQ ID NO: 72) K82 ACTCTGGAGCGTTCTC 1.9 0.1 38.5 5 25.5 16.810.7 0.6 (SEQ ID NO: 73) K30 TGCAGCGTTCTC 9.5 10.5 30.8 31 253.1 256.37.0 6.9 (SEQ ID NO: 74) k31 TCGAGGCTTCTC 22.1 26.3 30.7 18 290.3 150.612.9 9.0 (SEQ ID NO: 75) K39 GTCGGCGTTGAC 7.0 11.0 27.3 22 100.3 137.916.1 6.3 (SEQ ID NO: 76) K16 TCGACTCTCGAGCGTTCTC 3.1 3.1 25.2 21 72.779.5 22.3 20.6 (SEQ ID NO: 8) K3 ATCGACTCTCGAGCGTTCTC 9.0 22.7 22.6 17113.5 60.0 25.0 19.4 (SEQ ID NO: 3) k23 TCGAGCGTTCTC 5.2 8.3 21.9 28111.3 145.4 28.3 16.0 (SEQ ID NO: 79) DV110 TCGAGGCTTCTC 2.2 2.2 21.8 2570.3 142.8 19.6 19.8 (SEQ ID NO: 75) K40 GTCGGCGTCGAC 11.3 14.3 20.7 11127.1 222.2 11.0 5.0 (SEQ ID NO: 81) DV101 CTCGAGCGTTCT 2.6 2.2 20.0 2628.0 34.3 25.3 28.3 (SEQ ID NO: 10) DV89 ACTCTTTCGTTCTC 1.6 1.6 19.7 23169.1 390.3 6.7 7.2 (SEQ ID NO: 83) K34 GTCGACGTTGAC 4.3 5.3 17.2 8265.8 273.4 11.3 6.7 (SEQ ID NO: 84) DV86 ACT CTCGAGCGTTCTC 0.8 1.6 16.937 36.0 40.7 5.1 3.0 (SEQ ID NO: 9) K83 ACTCTCGAGGGTTCTC 0.0 0.0 16.5 516.9 9.7 6.9 0.7 (SEQ ID NO: 86) K19 ACTCTCGAGCGTTCTC 4.8 7.9 14.8 16151.0 223.6 23.4 16.1 (SEQ ID NO: 9) DV88 ACTCTCGAGCGTTCTCAAAA 1.9 3.014.5 14 49.7 55.0 10.2 11.5 (SEQ ID NO: 88) DV85 CATCTCGAGCGTTCTC 0.91.5 12.0 11 82.0 126.8 12.2 13.0 (SEQ ID NO: 89) K73 GTCGTCGATGAC 4.55.2 11.7 7 99.2 110.7 17.7 13.2 (SEQ ID NO: 90) DV109 TCGAGCGTTCT 2.22.2 11.3 9 28.4 46.4 14.5 12.6 (SEQ ID NO: 4) D123 TCGTTCGTTCTC 3.3 1.69.6 12 50.9 72.3 39.3 30.5 (SEQ ID NO: 92) D124 TCG TT TG TT CTC 3.0 2.07.3 6 63.2 88.6 34.1 28.1 (SEQ ID NO: 93) K46 GTCGACGCTGAC 3.5 5.1 5.2 221.9 15.1 8.7 5.0 (SEQ ID NO: 94) D139 TCGATGCTTCTC 2.2 1.4 5.1 4 97.8157.5 39.1 27.9 (SEQ ID NO: 95) D137 TCGCCGCTTCTC 1.4 0.5 4.3 2 78.7107.6 31.6 22.3 (SEQ ID NO: 96) K47 GTCGACGTCGAC 3.8 7.1 3.9 1 14.1 6.25.8 2.3 (SEQ ID NO: 54) K72 GTCATCGATGCA 1.8 2.3 3.7 3 95.6 79.3 11.05.4 (SEQ ID NO: 98) DV90 ACTCTTTCGATCTC 1.6 2.0 3.6 4 19.2 24.8 4.6 5.7(SEQ ID NO: 99) K37 GTCAGCGTCGAC 10.8 19.5 3.4 1 52.4 63.0 6.4 2.2(SEQ ID NO: 100) k25 TCGAGCGTTCT 3.6 3.7 2.9 4 17.9 24.8 (SEQ ID NO: 4)D127 TGG AG CG TT CTC 2.3 1.6 2.8 3 89.7 136.4 21.4 11.9(SEQ ID NO: 102) D138 TGCTGCGTTCTC 0.7 0.7 2.1 1 69.0 105.5 18.4 12.1(SEQ ID NO: 103) D125 TTG AG CG TA CTC 3.3 3.2 2.0 1 35.1 53.8 7.8 5.2(SEQ ID NO: 104) D134 TGC TT CGAGCTC 1.8 0.8 1.8 1 17.7 24.8 9.1 5.7(SEQ ID NO: 105) D136 TGCACCGTTCTC 1.7 0.2 1.7 1 69.2 106.5 12.1 7.9(SEQ ID NO: 106) CONTROL K ODN DV89 ACTCTTTCGTTCTC 1.6 1.6 19.7 23 169.1419.7 6.7 7.2 (SEQ ID NO: 83) d112 TGCAGGCTTCTC 16.0 28 20.4 32.6(SEQ ID NO: 6) DV112 TTGAGTGTTCTC 2.1 2.7 16.0 28 20.4 32.6 9.8 10.8(SEQ ID NO: 109) DV112 TTGAGTGTTCTC 2.1 2.7 16.0 28 20.4 32.6 9.8 10.8(SEQ ID NO: 109) K41 GTCGGCGCTGAC 5.7 7.4 13.3 8 29.1 24.7 8.8 5.7(SEQ ID NO: 120) DV109 TCGAGCGTTCT 3.4 4.0 11.3 9 28.4 46.4 14.5 12.6(SEQ ID NO: 4) k10 ATGCACTCTGCAGGCTTCTC 2.7 4.5 6.1 7 27.3 28.8 5.7 5.4(SEQ ID NO: 119) K38 GTCAGCGCTGAC 1.4 1.9 4.5 3 4.5 1.3 2.2 1.1(SEQ ID NO: 118) k29 TCGAGCG 19.7 25.9 4.2 5 2.8 3.6 (SEQ ID NO: 112)k26 TCGAGCGTTC 6.6 4.9 4.1 1 22.3 24.4 (SEQ ID NO: 45) k27 TCGAGCGTT 4.63.4 3.7 5 4.1 3.7 (SEQ ID NO: 108) K36 GTCAACGCTGAC 2.2 3.3 3.4 2 8.62.0 5.5 2.8 (SEQ ID NO: 107) K35 GTCAACGTCGAC 21.8 41.9 3.3 2 15.9 13.95.0 2.8 (SEQ ID NO: 101) K44 GTCGACGCCGAC 4.1 6.0 3.0 2 9.4 6.3 2.6 1.2(SEQ ID NO: 97) k28 TCGAGCGT 21.5 26.2 2.9 3 6.7 9.1 (SEQ ID NO: 91)AA19 GGGGGAACGTTGGGGG 9.4 14.6 2.2 1 4.7 4.5 1.5 0.5 (SEQ ID NO: 87)D135 TGCAGCGAGCTC 1.4 0.5 1.8 2 4.3 6.6 3.4 1.5 (SEQ ID NO: 85) D141CCGAGGCTTCTC 1.3 0.4 1.7 2 53.7 99.0 12.8 9.0 (SEQ ID NO: 82) D126 ACGAG GG TT CTC 2.6 0.8 1.7 1 75.5 107.5 15.2 8.7 (SEQ ID NO: 80) K42GTCAACGCCGAC 2.6 3.1 1.7 1 5.7 5.3 4.3 3.7 (SEQ ID NO: 78) D140GCGAGGCTTCTC 1.2 0.5 1.5 1 56.7 85.4 10.9 7.0 (SEQ ID NO: 77) d121ACTCTTGAGTGTTCTC 3.6 6.2 1.0 1 10.2 18.5 17.3 44.5 (SEQ ID NO: 66) K45GTCGGCGCCGAC 5.7 6.6 0.4 1 18.3 22.1 10.1 4.3 (SEQ ID NO: 57) K43GTCAGCGCCGAC 2.1 1.6 0.3 1 6.9 8.6 5.1 5.7 (SEQ ID NO: 56) K24CGAGCGTTCTC 4.9 5.1 0.1 0 0.7 1.0 1.0 0.1 (SEQ ID NO: 48) The foregoingdata demonstrates the induction of an immune response in human cells, asexemplified by PBMC. Specifically, the results demonstrate that K typeoligodeoxynucleotides induce IL-6, cell proliferation and a humoralresponse, while D type oligodeoxynucletides induce the production ofIFN-γ.

Example 2 Production of IL-6

The following example demonstrates induction of an immune response, asmeasured by cytokine production, by K type oligodeoxynucleotides.Specifically, it is demonstrated that production of the cytokine IL-6can be induced by K type oligodeoxynucleotides, and that B cells arestimulated by K type oligodeoxynucleotides.

A human B cell line (RPMI 8226) was maintained according to themanufacturers recommendations. ODNs were synthesized as described inExample 1. In some ODNs, the normal DNA phosphodiesterase linkages werereplaced with phosphorothioate linkages, as described in Example 1. Toreduce degradation of the ODNs, those that did not have an entirephosphorothioate backbone contained phosphorothioate linkages at theends. The cells were incubated with various ODNs for 14 hrs. IL-6production was determined by ELISA using anti-IL-6 antibodies, asdescribed in Example 1.

IL-6 levels are set forth in Table 1. These data confirm that a sequencecontaining 5′ N₁N₂N₃T-CpG-WN₄N₅N₆ 3′, which are linked byphosphorothioate bonds and wherein the central CpG motif isunmethylated, W is A or T, and N₁, N₂, N₃, N₄, N₅, and N₆ are anynucleotides, is desirable to induce a humoral immune response.

The foregoing data demonstrates the induction of an immune response inhuman cells by K type oligodeoxynucleotides, as exemplified by the humanB cell line RPMI 8226, and as measured by production of the cytokineIL-6.

Example 3 Exemplary Material and Methods

The following example delineates material and methods that were used toinvestigate the induction of an immune response by D-type ODNs.Exemplary D type ODNs are shown in Table 2 below.

TABLE 2 UNDERLINED = PHOSPHODIESTER; *METHYLATED C IFNg ODN AVG SD Media0.9 0.8 DV104 GGTGCATCGATGCAGGGGGG 47.7 64.7 (SEQ ID NO: 1) DV19GGTGCATCGATGCAGGGGGG 44.0 71.8 (SEQ ID NO: 1) DV29 GGTGCACCGGTGCAGGGGGG35.9 38.9 (SEQ ID NO: 2) DV35 GGTGCATCGATGCAGGGGGG 33.3 63.6(SEQ ID NO: 1) DV28 GGTGCGTCGATGCAGGGGGG 32.2 45.3 (SEQ ID NO: 7) DV106GGTGTGTCGATGCAGGGGGG 29.9 71.4 (SEQ ID NO: 26) DV116 TGCATCGATGCAGGGGGG25.1 28.9 (SEQ ID NO: 12) DV113 GGTGCATCGATACAGGGGGG 21.1 15.3(SEQ ID NO: 11) DV34 GGTGCATCGATGCAGGGGGG 16.2 19.1 (SEQ ID NO: 1) DV102GGTGCATCGTTGCAGGGGGG 15.8 39.4 (SEQ ID NO: 30) DV32 GGTGCGTCGACGCAGGGGGG14.5 18.0 (SEQ ID NO: 31) DV117 GGTCGATCGATGCACGGGGG 13.4 14.6(SEQ ID NO: 32) dv37 GGTGC ATCGAT GCAGGGGGG 13.0 19.6 (SEQ ID NO: 1)DV25 GGTGCATCGATGCAGGGGGG 12.2 7.9 (SEQ ID NO: 1) DV30GGTGCATCGACGCAGGGGGG 11.3 8.1 (SEQ ID NO: 35) dv120 GGTGCATCGATAGGCGGGGG8.7 11.5 (SEQ ID NO: 36) DV27 GGTGCACCGATGCAGGGGGG 8.5 7.3(SEQ ID NO: 37) dv119 CCTGCATCGATGCAGGGGGG 7.5 9.1 (SEQ ID NO: 38) D142GGTATATCGATATAGGGGGG 7.3 1.1 (SEQ ID NO: 39) d143 GGTGGAT CG ATCCAGGGGGG6.4 4.5 (SEQ ID NO: 40)

Normal PBMC were obtained from the National Institutes of HealthDepartment of Transfusion Medicine (Bethesda, Md.). The human myelomacell line RPMI 8226 (CCL-155; American Type Culture Collection,Ma-nassas, VA) and the NK-92 human NK cell line (a kind gift of Dr. J.Ortaldo, National Cancer Institute, Frederick, Md.) were grown in RPMI1640 supplemented with 10% FCS, penicillin/streptomycin, L-glutamine,HEPES, sodium pyruvate, and 2-ME in a 5% CO2 in-air incubator. Mediumfor NK-92 cells was supplemented with IL-2 (200 IU/ml; R&D Systems,Minneapolis, Minn.) and IL-15 (15 ng/ml; Endogen, Boston, Mass.).

Oligodeoxynucleotides

ODN were synthesized at the Center for Biologics Evaluation and Researchcore facility. All had <0.1 endotoxin U/ml endotoxin at ODNconcentrations of 1 mg/ml.

Antibodies

Abs against human IFN-g (Endogen), IL-6 (R&D Systems), and IgM(Se-rotec, Oxford, U.K.) were used for ELISA and enzyme-linkedimmunospot (ELISPOT) assays. FITC- and/or CyChrome-labeled Abs againsthumanCD3, CD4, CD14, CD11c, CD16, CD56, CD83, HLA-DR, IL-6, and IFN-γwere obtained from BD PharMingen (San Diego, Calif.) or BD Bio-sciences(San Jose, Calif.) and used as recommended by the manufacturer.Neutralizing Abs to IL-12 were obtained from R&D Systems, and Abs toIL-18 were kindly provided by Dr. Howard Young (National CancerInstitute).

Mononuclear Cell Preparation

Mononuclear cells were separated by density gradient centrifugation overFicoll-Hypaque as described (17). Cells were washed three times andcultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCSfor 72 h at 53 10 5 cells/well in the presence of 1-3 mM ODN.

ELISA and ELISPOT Assays

Ninety-six-well microtiter plates (Millipore, Bedford, Mass.) werecoated with anti-cytokine Ab or anti-IgM and blocked with PBS-5% BSA(17,18). Cytokines and Ig in culture sups or secreted by individualcells were detected colorimetrically using biotin-labeled Abs followedby phosphatase conjugated avidin and then phosphatase-specificcolorimetric substrate. Standard curves were generated to quantitateELISA results using known amounts of recombinant cytokine or purifiedIgM. The detection limit of the assays was: 6 pg/ml for IFN-g, 20 pg/mlfor IL-6, and 10 ng/ml for IgM. Stimulation index was calculated by theformula: (value for stimulated cells 2 background)/(value forunstimulated cells 2 background). In cases where cytokine/Ig productionwas below assay sensitivity, the lower limit of detection was used tocalculate the stimulation indices. All assays were performed intriplicate.

Proliferation Assays

A total of 10 5 PBMC/well were incubated with 3 mM of ODN for 68 h,pulsed with 1 mCi of [³H]thymidine, and then harvested 4 h later. Theproliferation index represents the fold difference between stimulatedand unstimulated cells. All assays were performed in triplicate.

Intracellular Cytokine Staining and Flow Cytometry

PBMC were cultured for 8 h (K type) or 24 h (D type) with 3 mM ofvarious ODN. Brefeldin A (20 mg/ml) was added to the cultures after 2 or12 h, respectively. Cells were harvested with warm PBS-0.02% EDTA andwashed. PBMC (1 3 10 6/sample) were fixed and permeabilized using theFix & Perm cell permeabilization kit (Caltag, Burlingame, Calif.) asrecommended by the manufacturer. Cells were then stained withPE-conjugatedanti-IL-6 or anti-IFN g plus specified FITC- or CyChrome-conjugated Abs against cell surface markers for 30 min indarkness. After labeling, the cells were washed twice, and 40,000 eventsper sample were analyzed by FAC-Scan flow cytometry (BD Biosciences).Cell Quest software (BD Bio-sciences) was used for data analysis.

Statistical Analysis

Statistically significant differences were determined using a two-tailnon-parametric Mann-Whitney U test and nonparametric ANOVA.

Example 4 Response of Human Peripheral Blood Mononuclear Cells (PBMC) toCpG ODN

Response of human PBMC to CpG ODN Novel ODN were studied for theirability to stimulate human PBMC to proliferate and/or secrete Ig orcytokines. As shown in FIG. 1, two structurally distinct ODN classeswere identified that stimulated PBMC from 0.95% of the donors. Those ofthe K type stimulated significantly greater cell proliferation(p<0.0001) and induced higher levels of IL-6 (240 vs 85 pg/ml; p <0.01)and IgM (695 vs 20 ng/ml; p<0.0001) than D ODN. In contrast, D ODN werestronger inducers of IFN-g (70 vs 13 pg/ml; p<0.05). FIG. 1 illustratesthe response of PBMC to K and D ODN.

Type D ODN

Modifications were introduced in various regions of D ODN to identifythe critical sequences and structures that account for the ability ofthese ODN to induce IFN-γ. To standardize results from the large numberof subjects and experiments included in the analysis, the magnitude ofeach response is presented as fold increase over cells from the samesubject incubated in medium alone. The general magnitude of theseresponses was comparable to that shown in FIG. 1.

FIG. 2 illustrates the parameters governing D ODN induced immuneactivation. All D-type ODNs contain an unmethylated CpG dinucleotide(FIG. 2). Inversion, replacement, or methylation of the CpG reduces orabrogates reactivity (FIG. 2A, line 1 vs lines 2-6, and line 7 vs line8; p<0.0001). In addition, the results demonstrate that D ODN arestimulatory only if the CpG dinucleotide and its immediate flankingregions are composed of phosphodiester (shown in gray) rather thanphosphorothioate nucleotides (FIG. 2B, line 1 vs line 2; p<0.001).Because phosphorothioate-modified nucleotides confer resistance toexonuclease digestion, they are incorporated at the ends of the ODN toimprove activity (FIG. 2B, lines 1 and 5 versus lines 3 and 4; p<0.07).Unless otherwise stated, all D ODN studied werephosphorothioate/phosphodiester chimeras.

As shown in FIG. 2, the level of immune stimulation induced by D ODN wasdemonstrated to be influenced by the bases flanking the CpGdinucleotide. Self-complementary hexamers consisting of a Pu Py CG Pu Pywere the most active, as exemplified by ATCGAT and ACCGGT (FIG. 2C,lines 1 and 2). Substituting a Pu for a Py, or vice versa, significantlyreduced or eliminated ODN activity (circled nucleotides in FIG. 2C,lines 1 and 2 vs lines 6-13; p<0.0001. By comparison, hexamers thatmaintained the PuPyCGPuPy sequence, but are non self-complementaryinduced lower levels of IFN-g production (FIG. 2C, lines 1 and 2 vslines 3-5; p<0.001). Sequential deletion experiments showed that theminimum length of an active D ODN is 16 bp (FIG. 2D; p<0.01). Thisfinding suggested that sequences outside the central hexamer mightinfluence D ODN activity.

Indeed, stimulation was maximal when the three bases on each side of theCpG-containing hexamer formed a self-complementary sequence (FIG. 2E,lines 1 and 2 versus lines 3 and 4; p<0.0001). Without being bound bytheory, computer modeling of D ODN suggested that theseself-complementary base sequences help form a stem-loop structure withthe CpGdi nucleotide at the apex at 37° C. The ends of the ODN alsocontribute to its activity, with the inclusion of more than four Gs atthe 39 end significantly increased function (FIG. 2F, lines 1-3 versuslines 4 and 5; p<0.001). Thus, modifications in any of the three areas(the central hexamer, the region flanking the hexamer, or the poly Gtail) can be used to influence ODN activity.

Type K ODN

K ODN trigger cell proliferation and the secretion of IgM and IL-6, butlittle IFN-g (see Table 1 and FIG. 1). These ODN have a phosphorothioatebackbone and at least one unmethylated CpG dinucleotide.

TABLE 5 Rules Governing K ODN Induced Immune ActivationStimulation index IL-6 Proliferation IgM Assay Subject #: 1 2 1 2 1 2A: Multiple CpGs induce more stimulation 1 ATCGACTCTCGAGCGTTCTC 50 57 1330 138 71 (SEQ ID NO: 3) 2 TCGAGCGTTCTC 35 40 15 37 19 79(SEQ ID NO: 79) 3 TCGAGGCTTCTC 28 12 8 25 8 22 (SEQ ID NO: 75) 4TGCAGGCTTCTC 1 0 5 7 5 4 (SEQ ID NO: 6)B: Minimum size of stimulatory “K” ODN 1 TCGACTCTCGAGCGTTCTC 20 23 3772 >100 100 (SEQ ID NO: 8) 2 ACTCTCGAGCGTTCTC 20 18 30 46 >100 100(SEQ ID NO: 9) 3 TCTCGAGCGTTCTC 16 40 28 41 >100 100 (SEQ ID NO: 72) 4TCGAGCGTTCTC 23 15 13 25 >100 80 (SEQ ID NO: 79) 5 CTCGAGCGTTCT 21 14 1621 95 92 (SEQ ID NO: 10) 6 TCGAGCGTTCT 10 4 15 35 45 78 (SEQ ID NO: 4) 7TCGAGCGTTC 6 5 17 40 35 82 (SEQ ID NO: 116) 8 TCGAGCGTT 1 7 5 7 32 25(SEQ ID NO: 117) 9 TCGAGCGT 1 5 1 13 25 12 (SEQ ID NO: 91) 10 TCGAGCG 11 1 5 9 4 (SEQ ID NO: 115) C: CpG motifs located at the 5′ end of the ODN are most stimulatory 1 TCGAGCGTTCTC 12 40 52 59 80 >100(SEQ ID NO: 79) 2 T CG AGGCTTCTC 6 12 51 61 >100 >100 (SEQ ID NO: 75) 3TGCTT CG AGCTC 4 3 12 16 20 60 (SEQ ID NO: 105) 4 G CG AGGCTTCTC 5 12 1814 >100 >100 (SEQ ID NO: 121) 5 TGCAG CG AGCTC 5 2 4 4 1 16(SEQ ID NO: 85) 6 CGAGCGTTCTC <1 <1 1 1 <1 2 (SEQ ID NO: 48)D. Optimization of the 5′ CpG flanking region 1 T CGATGCTTCTC 5 12 60 67100 9 (SEQ ID NO: 95) 2 T CGAGGCTTCTC 6 12 51 61 160 5 (SEQ ID NO: 75) 3A CGAGGCTTCTC 3 3 18 23 110 11 (SEQ ID NO: 34) 4 G CGAGGCTTCTC 3 1 18 1472 6 (SEQ ID NO: 127) 5 C CGAGGCTTCTC 4 1 15 25 25 7 (SEQ ID NO: 82) 6TGCTT CGAGCTC 3 1 12 16 60 40 (SEQ ID NO: 105) 7 TGCAG CGAGCTC 2 1 4 416 8 (SEQ ID NO: 82) E. Optimization of the 3′ CpG flanking region 1 TCG TTTGTTCTC 8 8 28 31 >100 >100 (SEQ ID NO: 93) 2 T CG TATGTACTC 8 8 2632 2 33 (SEQ ID NO: 33) 3 T CG GATGAGCTC 6 8 9 20 28 41 (SEQ ID NO: 29)4 T CG AATGCTCTC 3 5 14 22 6 14 (SEQ ID NO: 28) 5 TTGTT CG TTCTC 3 4 1514 19 26 (SEQ ID NO: 27) 6 TTGTT CG TACTC 2 4 14 13 15 72(SEQ ID NO: 25) 7 TTGTT CG AGCTC 2 4 6 4 6 16 (SEQ ID NO: 24) 8 TTGTT CGAACTC 2 2 11 5 1 1 (SEQ ID NO: 24) Over 200 novel ODN were synthesized,and their ability to activate PBMC from multiple donors examined. PBMCwere stimulated for 72 hours in the presence of ODN (1 μM added at time0). Cytokine and antibody secretion in the supernatants were assessed byELISA, while proliferation was determined by [H]³ uptake. Examples ofgeneral findings are presented in this Table. Results are expressed asfold increase over unstimulated cells.

As with D ODN, eliminating the CpG dinucleotide from a K type ODNsignificantly reduced immune activation (Table 3A, line 3 versus line 4;p, 0.02). Incorporating multiple CpGs in a single ODN increased immunestimulation (Table 3A, lines 1-3). To determine the minimum length of astimulatory K ODN, nucleotides were sequentially deleted from each end.ODN at least 12 bases long consistently induced strong immune cellactivation, whereas shorter ODN were relatively less active (Table IB,lines 1-5 vs lines 6-10). CpG motifs at the 5′ end were the moststimulatory (Table 3C, line 2 vs line 3 and line 4 vs line 5), althoughat least one base upstream of the CpG was required (Table 3C, line 1 vsline 6). Indeed, a thymidine in the immediate 5′ position (Table 3D,lines 1 and 2 vs lines 3-5 and line 6 vs line 7) and a 3′ TpT or a TpA(Table 3E, lines 1 and 2 vs lines 3 and 4 and lines 5 and 6 vs line 7)yielded the most active K ODN. Modifications >2 bp from the CpGdi-nucleotide had relatively less effect on ODN activity.

Example 5 Cellular Targets of K and D ODN

The phenotype of the cells stimulated to produce cytokine was determinedby combined cell surface and intracytoplasmic staining. As seen in Table4, D ODN selectively stimulated CD3-CD16+CD56+CD14-cells to produceIFN-γ, consistent with the direct activation of NK cells.

TABLE 4 Phenotype of PBMC stimulated by CpG ODN to secrete cytokinesCell surface marker % positive cells A. Phenotype of PBMC activated by“D” ODN to produce IFNγ CD16 91 CD56 99 CD3 5 CD14 <1 B. Phenotype ofPBMC activated by “K” ODN to produce IL-6 CD83 93 CD14 70 CD11c 69 CD1913 CD16 <1 Freshly isolated PBMC were stimulated with K” (8 hr) or D (24hr) ODN. Cells were fixed, permeabilized and stained with PE- anti-IL-6or anti-IFNγ. Results are representative of 4-10 experiments.

Without being bound by theory, the effect appears to be direct because DODN do not induce a significant increase in IL-12 secretion. Moreover,studies using neutralizing anti-IL-12, which reduce the production ofIFN-g by PBMCs stimulated with PHA (44% p, 0.05) or with bacillusCalmette-Guerin (77%; p, 0.05), did not decrease the IFN-γ productioninduced by CpG-ODN.

By comparison, K ODN stimulated CD14+, CD11c+, and CD83+ cells toproduce IL-6, indicating that they were of monocyte/dendritic celllineage (Table 4B). K ODN also stimulated a fraction of CD19+ B cells torelease IL-6.

To confirm these findings, human NK, T, and B cell lines were tested fortheir responsiveness to K and D ODN. The NK-92 cell line respondedexclusively to D ODN by secreting IFN-γ, whereas the human RPMI 8226 Bcell line was stimulated by K ODN to release IL-6. Non-CpG ODN did notstimulate either cell line.

Thus, as disclosed herein, there are two structurally distinct types ofCpG ODN that stimulate different cellular elements of the immune systemto mount divergent responses. K type ODN induce monocytes/dendriticcells to produce IL-6 and B cells to proliferate and secrete IgM,whereas D type ODN support NK production of IFN-γ (see Table 1). TheseCpG ODN can be used as vaccine adjuvants, anti-allergens, andimmunoprotective agents, as disclosed herein.

The CpG motifs at the center of K and D ODN differ. The optimal K motifcontains a thymidine immediately 5′ of the CpG, and a TpT or TpA 3′ ofthe CpG. By comparison, optimally active D type ODN contain Pu-Py dimerson each side of the CpG. D ODN also are longer. In one embodiment, theCpG flanking regions are self-complementary. Two dimensional computermodeling further suggested that this self-complementary sequencefacilitates the formation of a hairpin loop that exposes the CpG at theapex. Without being bound by theory, this stem-loop structurecontributes to the recognition of D ODN because IFN-γ productiondeclines when the length or binding strength of the palindrome isreduced (FIG. 2E). Without being bound by theory, the inclusion of polyG at the 3′ end of the ODN likely further confers a structural benefit.Alternatively, the poly Gs may improve the efficiency of cellularuptake.

As demonstrated herein, K and D ODNs activate distinct cell types. K ODNactivate monocytes and B cells to secrete IL-6, whereas D type ODNstimulate NK cells to secrete IFN-γ (Table 1). It is believed that thisdifference is not due to differential uptake of the ODNs, as monocytesand NK cells take up both D and K ODNs. Without being bound by theory, Kand D type ODNs activate their target cells directly, as 1) CpG ODNstimulate cloned cell lines to secrete cytokines; 2) cytokine mRNAappears within minutes of ODN stimulation; and 3) ELISPOT studies showthat the CpG ODN induced rapid increase (two to five-fold) in IL-6 andIFN-γ secreting PBMC (5 and 18 hour after stimulation, respectively).Moreover, flow cytometric analysis of cells stimulated to secrete IFN-γby CpG ODN were CD3-even after 72 h of stimulation, indicating that theincreased IFN-γ in supernatents is not due to the secondary activationof T cells.

Example 6 Further Evidence of Differing Functions of D and K ODNsMaterial and Methods

Oligonucleotides and antibodies: ODN were synthesized at the CBER corefacility. Sequences of the CpG ODN used in this study are:5′-TCGAGCGTTCTC-3′ (K23, SEQ ID NO: 79) and 5′-GGtgcatcgatgcaggggGG-3′(D35, SEQ ID NO: 1). The control for K ODN was: 5′-TCAAGTGTTCTC-3′ (SEQID NO: 122) and for D ODN was: 5′-GgtgcatctatgcaggggGG-3′ (SEQ ID NO:123). In this example, bases shown in capital letters arephosphorothioate while those in lower case are phosphodiester. CpGdinucleotides are underlined. All FITC, PE and cychrome labeled Mabswere purchased from Pharmingen (San Jose, Calif.). All ODNs used in thisstudy contained <0.1 U/mg of endotoxin.

Cell cultures: PBMC from normal donors (provided by the NIH Departmentof Transfusion Medicine) were isolated by Ficoll-Hypaque densitygradient centrifugation (Verthelyi, D, et al., J. Immunol.166:2372-2377, 2000). Countercurrent centrifugal elutriation was used toisolate monocytes that were >95% pure. 0.5-4×10⁶ cells/ml were culturedin RPMI 1640 containing 5% FCS, 50 U/ml penicillin, 50 μg/mlstreptomycin, 0.3 mg/mL L-glutamine, 0.1 mM non-essential amino acids, 1mM sodium pyruvate, 10 mM HEPES and 10⁻⁵ M 2-mercaptoethanol. Cells werestimulated with ODN for 8-72 h with 1-3 μM ODN, depending upon theassay.

Analysis of cell proliferation: PBMC were cultured in complete mediumplus 3 μM ODN for 72 h. To study B cell proliferation, cells were loadedwith 10 nM of CFSE (Molecular Probes Inc., Eugene, Oreg.) as describedbefore (24). Proliferation of CD11c+ monocytes was monitored by adding10 μM of BrdU (Pharmingen, San Jose, Calif.) for the last 18 h ofculture. Staining for BrdU was performed as recommended by themanufacturers.

ELISA assays: 96-well microtiter plates (Millipore, Bedford, Mass.) werecoated with anti-cytokine or anti-IgM Ab and blocked with PBS-5% BSA(8). The plates were incubated for 2 h with culture supernatants fromPBMC (5×10⁵/ml) that had been stimulated for 8-24 h with ODN asdescribed above. IL-6, IFNγ and IgM were detected colorimetrically usingbiotin-labeled antibodies followed by phosphatase-conjugated avidin anda phosphatase specific colorimetric substrate (Verthelyi, D, et al., J.Immunol. 166:2372-2377, 2000). The detection limit of the assays was: 6pg/ml for IFNγ, 20 pg/ml for IL-6, and 10 ng/ml for IgM. All assays wereperformed in triplicate.

Staining for cell surface markers and Intracellular cytokine: Culturedcells were washed in cold PBS, fixed, and stained with fluorescentlabeled anti-CD69 (24 h), anti-CD25 (72 h), anti-CD83 (72 h) oranti-CD86 (72 h). To detect intracytoplasmic cytokine, cells incubatedwith ODN for 8 h were washed, fixed, permeabilized (as permanufacturer's instructions, Caltag, Calif.) and stained with 4 μg/mlPE-conjugated anti-IL-6 or 2 μg/ml PE-conjugated anti-IFNγ (Pharmingen,San Jose, Calif.) plus various FITC and Cy-Chrome labeled surfacemarkers for 30′ at RT. Samples were washed and analyzed (20,000-40,000events) on a FACScan flow cytometer (Becton Dickinson, San Jose, Calif.)after gating on live cells with proper electronic compensation. The datawere analyzed using CELLQuest software (Becton Dickinson ImmunocytometrySystems, San Jose, Calif.).

Analysis of cell-surface binding and internalization of ODN: PBMC(4×10⁶/ml) were incubated with biotinylated ODN (1-3 μM) for 10′ at 4°C. (binding experiments) or 37° C. for 1 h (uptake experiments). Todetect internalized ODN, surface bound ODN was blocked with 100 μg/ml of“cold” streptavidin. After washing, these cells were permeabilized,fixed, and stained with PE-conjugated streptavidin (1 μg/ml) plus FITCor Cy-Chrome conjugated cell surface markers.

Confocal Microscopy Elutriated monocytes (4×10⁶/ml) were incubated withCy-3 or FITC labeled K and/or D ODN at 37° C. for 1 h. The cells werewashed, and mounted using the Prolong antifade kit (Molecular Probes,Oregon, USA). Subcellular localization of Cy3 and FITC labeled ODN wasdetermined by confocal microscopy under 1000× magnification (LSM5PASCAL, Carl Zeiss In., Thornwood, N.Y.).

Binding and Internalization of CpG ODN.

The ability of human PBMC to bind and internalize CpG ODN was examinedusing fluorescent-labeled K23 and D35 ODN. Both types of ODN boundrapidly to the surface of virtually all human monocytes at 4° C. Asignificant fraction of B lymphocytes (20-45%) and NK cells (˜10-20%)also bound these ODN. Simultaneous staining with K and D ODN showed thatthe same cells were binding both types of ODN. In contrast, interactionwith T cells barely exceeded background levels.

To monitor internalization, PBMC were incubated with biotin-labeled ODNfor 60′ at 37° C. Surface-bound ODN was blocked with excess strepavidin,and internalized ODN detected by staining fixed, permeabilized cellswith FITC-avidin. The fraction of monocytes, B lymphocytes and NK cellsthat internalized K23 and D35 ODN was similar to the fraction of eachcell type that bound these ODN. Similar results were obtained usingother D and K ODN. No internalization was observed when cells wereincubated with ODN for 10′ at 4° C., suggesting that ODN uptake involvesmetabolic activity.

The ratio of membrane bound: internalized ODN was compared. Based ondifferences in mean fluorescence intensity, it was calculated thattarget cells internalized approximately half of the ODN that had boundto their cell surface. For all cell types, the absolute magnitude of DODN uptake exceeded that of K ODN. For example, the amount of labeled DODN that bound to and was taken up by monocytes exceeded that ofequimolar K ODN by ˜2-fold throughout the functional concentration rangeof these agents (p<0.001). To achieve equivalent levels of binding anduptake required that “D” ODN be used at a 4-fold lower concentrationthan K (e.g. 0.75 vs 3.0 μM).

The intracellular localization of these two types of ODN was examined byconfocal microscopy of labeled monocytes. K and D ODN largely occupieddiscrete areas within the same cell, although there was a limited degreeof co-localization. D ODN largely occupied punctuated vesicles, whereasK ODN were more diffusely distributed, staining the nucleus as well ascytoplasmic vesicles. This difference in localization was associatedwith the presence or absence of a poly G tail, since control (non-CpG)ODN with a poly G tail showed the same distribution pattern as did DODN. In contrast, the fluorescent dyes used did not influencedistribution, since switching dyes had no effect on ODN localizationpattern.

Differential Effect of K Versus D ODN on B Cell Function

Whole PBMC were treated with optimal concentrations of K23 and D35 ODN.K ODN rapidly activated CD19⁺ B cells, reflected by a significantincrease in the expression of the CD69 early activation marker and theCD25 late activation marker (p<0.001). K ODN also triggered a >10-foldincrease in B cell proliferation (p<0.05), a >10-fold increase in IgMproduction (p<0.01), and a 5-fold increase in the number of B cellssecreting IL-6 (p<0.001). The effect of K23 exceeded that of D35 (and ofa control for the K type ODN of the same structure but lacking thecritical CpG motif) by >10-fold in each of these functional assays. DODN were not entirely inactive, however, since they induced a modestincrease in CD25 and CD69 expression by CD19⁺ B cells.

Differential Effect of K Versus D ODN on NK Cells

NK cells were identified by their expression of the CD16 surface marker.D ODN stimulated approximately 25% of these cells to increase expressionof CD25 and CD69 (p<0.001). Consistent with previous studies, D ODN alsotriggered a significant increase in IFNγ secretion by NK cells (p<0.05).By comparison, neither K ODN nor a non-CpG control for the D ODNsignificantly stimulated IFNγ production (see also Table 1). K23 didinduce a modest increase in the number of NK cells expressing CD25 andCD69 (p<0.05). None of these ODN induced NK cells to proliferate.

Differential Effect of K Versus D ODN on Monocytes

K and D ODN had disparate effects on purified monocytes. K23 stimulatedCD14⁺ monocytes to proliferate (p<0.05) and secrete IL-6 (p<0.001),while D35 had no effect in these assays (Table 5). Instead, D (but notK) ODN stimulated monocytes to mature into CD83⁺/CD86⁺ dendritic cells(DC) (p<0.001, Table 5). The divergent effects of K versus D ODN onmonocytes persisted throughout the physiologic concentration range ofboth types of ODN, and was observed using a variety of D and K ODN,indicating that these differences were not due to variation in ODNbinding or uptake. Although both types of ODN increased CD69 and CD25expression, D ODN up-regulated these activation markers in monocytessignificantly more effectively (p<0.001, Table 5).

TABLE 5 Effects of D ODN % of Cells Expressing/Producing ODN type CD25CD 69 IL-6 L-6 (ng/ml) Proliferation (SI) DC maturation K23 32.8 ± 1.139.7 ± 5.3 24.1 ± 2.4  2.0 ± 0.3 7.0 ± 1.4 3.5 ± 0.3 D35 48.6 ± 7.5 82.1± 2.4 4.6 ± 0.3 0.8 ± 0.1 1.0 ± 0.5 23.7 ± 2.0  K23 + D35  44.7 ± 13.9 53.8 ± 12.5 ND 2.2 ± 0.1 2.8 ± 1.7 5.2 ± 0.8 “K” control 21.8 ± 2.230.2 ± 2.5 5.1 ± 0.6 <.03 2.1 ± 1.6 1.8 ± 0.1 PBMC or elutriatedmonocytes were stimulated with 3 μM of ODN for 8-72 h. The percent ofCD14⁺ monocytes induced to secrete IL-6 was determined byintracytoplasmic staining. Due to the down-regulation of CD14, CD11c wasused to monitor the expression of CD25 and CD69 by stimulated cells. Thepercent of CD83⁺/CD86⁺ dendritic cells in culture was determined after72 h. IL-6 levels in culture supernatants were determined by ELISA,while proliferation was evaluated by BrDU incorporation. Resultsrepresent the mean ± SD of 5 independent experiments.

Competition Between K and D ODN at the Single Cell Level

The above findings suggested that monocytes responded differently tostimulation by K versus D ODN. There are two possible explanations forthis observation: either i) these two types of ODN were triggering thesame cells to mount distinct types of immune response or ii) K and D ODNwere acting on different subpopulations of monocytes. The latterexplanation seemed unlikely, given that confocal microscopy showed thatthe same cells were binding and internalizing both types of ODN.

To clarify this situation, monocytes were treated simultaneously withD35 plus K23. At optimally stimulatory concentrations, these ODN did notcross-compete for uptake or binding. Yet when their function wasanalyzed, co-administration of K ODN reduced the ability of D ODN totrigger monocyte differentiation by 70% (p<0.001). The inhibitory effectof K ODN on the activity of D ODN was sequence specific andconcentration dependent, since control non-CpG ODN did not significantlyinterfere with the activity of D ODN. Conversely, D ODN significantlyreduced the ability of K ODN to induce monocytes to proliferate(p<0.05). As above, the inhibitory effect of D on the activity of K ODNwas sequence specific and concentration dependent.

A very different pattern emerged when B and NK cells were studied. Inthese cells, the co-administration of D with K ODN was not inhibitory.Rather, the ability of K ODN to stimulate B cells to proliferate andsecrete IL-6 and IgM was unaffected by the presence of D ODN, and theability of D ODN to stimulate NK cells to secrete IFNγ was not reducedby inclusion of K ODN.

Example 7 D ODN Enhance Tumor Lysis

Reports of tumor regression following systemic bacterial infectionstimulated research into immune adjuvants as a strategy for tumortreatment. Immunostimulatory oligodeoxynucleotides (ODNs) containing theCpG motif have been demonstrated to induce the production of variouscytokines, natural killer cells, monocytes, lymphocytes and dendriticcells (see above). Thus, short-term bladder transitional cell carcinoma(TCC) cultures were treated with CpG ODN immunostimulated PBMC and thecultures were analyzed for evidence of tumor lysis.

Materials and Methods:

Papillary TCCs from 4 patients were cultured. Peripheral bloodmononuclear cells (PBMCs) were obtained from each patient. In matchedcultures, the tumor cells were treated with PBMCs stimulated in-vitrowith: 1) CpG ODN in the presence or and absence of tumor cells, 2)bacillus Calmette-Guerin (BCG) stimulated PBMCs (positive control andstandard of care), 3) unstimulated PBMCs, and/or 4) BCG or CpG ODN inthe absence of the patient's PBMCs (negative controls).

The morphologic and lytic characteristics of these cultures werecompared with those of untreated matched tumor cell cultures. A chromiumbased CTL assay method was used for quantitative comparison of tumorlysis.

Results

PBMCs that were stimulated in vitro with CpG ODN type D in the presenceof tumor cells lysed 30-70% of the tumor cells from 3 of 4 patients.

TABLE 6 Percent lysis PBMC + TCC 4 + 5 PBMC + TCC + D ODN 41 + 15 PBMC +TCC + K ODN 3 + 3 PBMC + TCC + CONTROL ODN 3 + 2 PBMC + TCC + BCG 71 +24 Thus, CpG ODN immunostimulation was associated with enhancement oftumor lysis comparable to that of BCG. K ODN did not enhance tumorlysis. Similarly, tumor lysis was not enhanced in the absence of CpG ODN(the negative control).

Example 8 Primates Provide a Model System for the Study of CpG ODN

Primate PBMCs respond in a similar manner as human PBMCs to CpG ODN.

To demonstrate the similarities, PBMC from normal human donors andRhesus macaques were stimulated with a panel of K, D or control ODNs,and the IL-6 and IFN-levels produced were monitored (FIGS. 4 and 5). Inboth human and monkey PBMC cultures, D ODNs induced the secretion ofIFN-γ, while K ODNs induced cells proliferation and IL-6 production.Thus, rhesus macaques provide a accurate model system for studying theeffects of ODNs in vitro.

In order to establish that rhesus macaques were a model system for invivo experiments, macaques were treated an antigen (OVA) and with eitherD type (D19 and D29) or K type (K3 and K23) ODNs, or with a control ODN(A23). The anti-OVA IgG titers in the group that received D ODN weresignificantly higher (FIG. 6), thus demonstrating that D ODN canincrease the immune response to a protein antigen in vivo. Thus, rhesusmacaque monkeys provide an in vivo model system to study the effects ofODNs.

Example 9 Effect of D ODN on PBMC from Primates

As disclosed in this example, rhesus macaques provide a useful model forassessing the activity of CpG ODN in vivo. In vitro studies establishedthat PBMC from rhesus macaques responded to the same panel of K and DODN that were highly active on human PBMC. CpG ODN were co-administeredwith a mixture of ovalbumin plus alum. The ODN significantly boosted theantigen-specific IgG response of macaques, with D being superior to KODN. A cutaneous leishmania infection model was then used to examinewhether CpG ODN could boost protective immunity in primates. The nature,severity, duration and histopathology of the cutaneous disease caused byL. major in macaques is quite similar to that in humans (for examplesee. Amaral et al., Exp Parasitol 82:34, 1996). Results indicate that DODN significantly improve the protection conferred by co-administeredheat-killed leishmania vaccine (HKLV).

Materials and Methods Utilized

Rhesus monkeys: Healthy 3 year old female rhesus macaques (M. mulata)were obtained from the FDA colony in South Carolina. All studies wereACUC approved and were conducted in an AAALAC accredited facility.Animals were monitored daily by veterinarians. No systemic or localadverse reactions to CpG ODN, OVA or HKLV immunizations were observed.Treatments were administered and peripheral blood samples obtained fromketamine anesthetized animals (10 mg/kg, Ketaject, PhoenixPharmaceuticals, St Joseph, Md.).

Vaccination groups and protocol: Two in vivo studies were conducted. 1)3 monkeys/group were immunized subcutaneously (s.c.) and boosted 12weeks (wk) later with a mixture of 4 μg of ovalbumin, 250 μg ODN and 125μg of alum (Rehydragel HPA, Reheis, Berkeley Heights, N.J.). 2) 5-6monkeys/group were immunized s.c. and boosted 4 weeks later with 250 μgof GMP grade HKLV (Biobras, Montes Claros, Brazil) plus 125 μg of alum,as previously described (27). The HKLV was administered alone, orcombined with 500 μg of ODN. Preliminary studies established that thisdose of ODN was active in vivo and well-tolerated. Animals were exposedto non-viable L. amazonensis metacycle promastigotes on week 8, atreatment that induced no disease and no change in antibody titer orproliferative response to Leishmania antigens when compared to controlanimals. Animals were challenged on the forehead on week 14 with 10⁷viable L. major (WHOM/IR/-/173) metacyclic promastigotes i.d. Themonkeys developed a typical self-limited in situ lesion characterized byerythema, induration, and ulceration. Lesion size, which reflects theseverity of infection, was measured weekly.

Oligodeoxynucleotides: ODN were synthesized by the CBER Core Facility.All ODN had less than <0.1 EU of endotoxin per mg of ODN as assessed bya Limulus amebocyte lysate assay (QCL-1000, BioWhittaker). The followingODN were used in this work:

D19: GGTGCAT CG ATGCAGGGGGG (SEQ ID NO: 1) D29: GGTGCAC CG GTGCAGGGGGG(SEQ ID NO: 2) D35: GGTGCAT CG ATGCAGGGGGG (SEQ ID NO: 1) D122:GGTGCATTGATGCAGGGGGG (SEQ ID NO: 53) K3: AT CG ACTCT CG AG CG TTCTC(SEQ ID NO: 3) K123: T CG TT CG TTCTC (SEQ ID NO: 92) K23: T CG AG CGTTCTC (SEQ ID NO: 79) K163: T TG AG TG TTCTC (SEQ ID NO: 109) AA3M:GGGCATGCATGGGGGG (SEQ ID: 124)

Mononuclear cell preparation: Human and monkey mononuclear cells wereisolated by density gradient centrifugation of PBMC over Ficoll-Hypaqueas described (see above). Cells were washed 3 times and cultured in RPMI1640 supplemented with 10% heat-inactivated FCS (FCS), 1.5 mML-glutamine and 100 U/ml of penicillin/streptomycin at 5×10⁵ cells/wellin the presence of 3 μM ODN. Supernatants were collected after 72 hoursand tested by ELISA for cytokine and antibody levels.

ELISA: 96-well microtiter plates (Millipore Corp., Bedford, Mass.) werecoated with Abs that cross-reactively recognized human and macaque IL-6(R&D, Minneapolis, Minn.), IFN-α (PBL Biomedical Laboratories, NewBrunswick, N.J.), and IgG (Boehringer-Mannheim Biochemicals, Germany).The plates were blocked with PBS-5% BSA. Culture supernatants from PBMCcultures were added, and their cytokine content quantitated by theaddition of biotin-labeled anti-cytokine Ab followed byphosphatase-conjugated avidin and phosphatase-specific colorimetricsubstrate. Standard curves were generated using known amounts ofrecombinant human cytokine or purified Ig. All assays were performed intriplicate. Titers of antibodies to ovalbumin in sera were assayed onOVA-coated plates.

ELlspot: The number of PBMC secreting IFN-γ in response to solubleleishmania antigen (SLA) were determined by ELlspot as described(Hagiwara Cytokine 7:815, 1995). Briefly, Millipore 96-well filtrationplates (Millipore Corp., Bedford, Mass.) were coated overnight at 4° C.with 1 μg/ml of anti-human IFN-γ antibodies (Clone GZ4, Alexis, SanDiego, Calif.) in PBS and then blocked with PBS-5% BSA for 2 hr. Theplates were overlaid with 5×10⁵ cells/well (1-2 series/monkey) andincubated at 37° C. in a humidified 5% CO₂ in air incubator for 18 hr inthe presence of 25 μg soluble leishmania antigen (SLA). The plates werethen washed with water-0.025% Tween and overlaid with biotin conjugatedanti-human IFN-γ (clone 76-B-1, Mabtech, Sweden). After 2 hr the plateswere washed again and then overlaid with alkaline phosphatase-conjugatedstreptavidin. Spots were visualized by the addition of5-bromo-4-chloro-3-indolyl phosphate (Kirkegaard and Perry Laboratories,Gaithersburg, Md.) and counted using the KS ELIspot Imagine System (CarlZeiss, Inc., Thornwood, N.Y.).

Cell proliferation assay: 10⁵ PBMC/well were incubated with 3 μM of ODNfor 68 h, pulsed with 1 μCi of [³H] thymidine and harvested 4 h later.All assays were performed in triplicate.

Statistical Analysis: Statistically significant differences weredetermined using a 2-tailed non-parametric ANOVA with Dunnett's posttest analysis. Differences in lesion sizes were tested byrepeated-measures ANOVA using the Proc Mixed procedure from theStatistical Analysis System (SAS).

Results:

Response of PBMC from human and non-human primates to K and D ODN

As disclosed herein, human PBMC respond to two structurally distinctclasses of CpG ODN. D type ODN triggered the secretion of IFN-γ andIFN-α whereas K ODN induced human PBMC to proliferate and secrete IL-6and IgM (Table 1). D and K ODN that strongly activated human cells weretested for their ability to stimulate PBMC from rhesus macaques.

The response of rhesus PBMC to “D” ODN was evaluated on the basis ofIFN-γ production. Results show that macaque PBMC are activated by thesame D ODN that stimulate human PBMC (p<0.002, FIG. 4). In contrast,neither K ODN, nor control ODN that are structurally similar to D ODNbut lack the critical CpG dinucleotide, had this effect.

Proliferation and IL-6 secretion were used to compare the response ofmacaque and human PBMC to K ODN. PBMC from both species were stimulatedby K ODN to proliferate (p<0.002) and secrete IL-6 (p<0.01), whereascontrols of the same structure as K ODN but lacking the critical CpGmotif failed to trigger immune stimulation. The results demonstratedthat the pattern of reactivity of PBMC from rhesus macaques (N=20) andhumans (N=8-20) to K and D ODN is similar.

Mixtures of ODN were identified that strongly stimulated PBMC from allhuman donors were utilized in further experiments. These mixtures weretested on PBMC from macaques and found to be uniformly active (FIG. 5).Subsequent in vivo studies were conduced with these ODN mixtures.

Adjuvant Activity of CpG ODN In Vivo

Previous studies in mice showed that CpG ODN could boost the immuneresponse to a co-administered protein antigen (such as ovalbumin). Thiseffect was amplified by adding alum to the mixture of CpG ODN plusantigen (for example see Klinman, Vaccine 17:19, 1999). Building onthese results, macaques were immunized and boosted with a mixture ofOVA, alum, and ODN (see FIG. 6). Animals immunized with mixturescontaining DODN increased their IgG anti-OVA response by 470-fold afterprimary (p<0.05) and by 600-fold after secondary (p<0.01) immunization.By comparison, K ODN boosted the IgG Ab response by 7-fold after primaryand 35-fold after secondary immunization when compared to pre-treatmentvalues (p<0.05). Macaques immunized with OVA plus control ODN generatedonly a 4-fold increase in anti-OVA titer. These findings indicate that DODN are particularly effective at boosting the antigen-specific humoralresponse to a co-administered antigen.

CpG ODN that activate human immune cells in vitro are only weaklyimmunostimulatory in mice. The results disclosed herein document thatrhesus macaques are a relevant model for examining the in vivo activityof CpG ODN. PBMC from macaques mirrored the response of human PBMC intheir response to both “K” and “D” ODN. At the immunostimulatory dosesused in this study, neither type of ODN triggered any adverse events. Invivo, broadly immunostimulatory mixtures of K and D ODN boostedantigen-specific IgG responses in macaques immunized with OVA andincreased IFN-γ production in animals vaccinated with HKLV. In addition,as described below, D ODN significantly increased the protectiveresponse elicited by a co-administered vaccine.

Example 10 Effect of CpG ODN on the Immunogenicity and ProtectiveEfficacy of Heat-Killed Leishmania Vaccine (HKLV)

Previous human clinical trials showed that HKLV was safe, but poorlyimmunogenic (Handman et al., Clin Microboiol. Rev. 14:229, 2001). Inaddition, HKLV combined with alum and IL-12 induces short-termprotective immunity in rhesus macaques (Kenney et al., J Immunol163:4481, 1999), and CpG ODN plus alum increased the immune response tothe hepatitis B vaccine in cyalomongus monkeys (Hartmen et al., JImmunol 164:1617, 2000).

Macaques were immunized and boosted with a mixture of HKLV, alum and CpGODN. PBMC from these animals were isolated 10 days post boost andre-stimulated in vitro with leishmania antigen for 72 hr. As seen inFIG. 7, both K and D ODN significantly increased the number of PBMCtriggered to secrete IFN-γ (p<0.05). In contrast, animals immunized withalum-adsorbed HKLV alone showed no increased IFN-γ production whencompared to unimmunized controls.

The critical measure of an antigen/adjuvant combination is its abilityto induce protective immunity. Vaccinated animals were thereforechallenged with 10⁷ L. major metacyclic promastigotes. Animalsvaccinated with HKLV-alum alone developed typical cutaneous lesions witha peak surface area of 300+60 mm² 26 days after challenge (FIG. 7).Monkeys vaccinated with HKLV-alum plus K ODN developed lesions ofsimilar size, although the peak lesion formation was slightly delayed.Animals immunized with HKLV-alum plus “D” ODN had significantly smallerlesions (maximal size 80+13 mm², p<0.05), consistent with a reducedparasite burden.

All animals treated with CpG ODN, either alone or with antigen, remainedhealthy and active throughout the study. No hematologic or serologicabnormalities were observed 3 days or 9 months after injection and noweight loss or change in behavior were detected following administrationof CpG ODN at therapeutic doses

As disclosed herein, cutaneous infection of macaques with L. majorprovides a means for testing the protective efficacy of CpG ODN-vaccinecombinations. The nature, severity and duration of the cutaneous diseasecaused by L. major in macaques is quite similar to that in humans. Theleading leishmania vaccine candidate (HKLV) has proven safe but poorlyimmunogenic in clinical trials (Handman, Clin Microboiol. Rev. 14:229,2001). Co-administration of both D and K ODN with this alum-adjuvantedHKLV vaccine significantly increased the number of PBMC triggered tosecrete IFN-γ when stimulated with leishmania antigen in vitro. However,the critical test of any vaccine/adjuvant combination is its ability toinduce protective immunity. Results show that the cutaneous lesions ofmacaques vaccinated with HKLV plus “D” ODN were significantly reducedwhen compared to HKLV-alum alone. A reduction in lesion size correlateswith a reduced parasite load. Thus, without being bound by theory, thefindings suggest that the ability of D ODN to stimulate IFN-γ and IFN αproduction while promoting the maturation of antigen presenting cellscan be useful for the induction of a protective response againstleishmania. In addition, without being bound by theory, the findingssuggest that the ability of D ODN to stimulate IFN-γ and IFN αproduction while promoting the maturation of antigen presenting cellscan be useful as a vaccine adjuvant.

All of the references cited herein, including patents, patentapplications, and publications, are hereby incorporated in theirentireties by reference.

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations of the preferred embodiments may be used and it isintended that the invention may be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications encompassed within the spirit and scope of the inventionas defined by the following claims.

1. An isolated stabilized D-type oligodeoxynucleotide of no more than 30nucleotides in length comprising the nucleic acid sequence set forth as5′XXTGCGCCGATGCAGGGGGG3′; (SEQ ID NO: 18) 5′XXTGCATCGACGCAGGGGGG3′;(SEQ ID NO: 19) 5′XXTGCGTCGGTGCAGGGGGG3′; (SEQ ID NO: 20)5′GGTGCATCGTTGCAGGGGGG3′; (SEQ ID NO: 30) or 5′ GGTGGATCGATCCAGGGGGG.(SEQ ID NO: 40)


2. The isolated stabilized D-type oligodeoxynucleotide of claim 1,wherein the oligodeoxynucleotide comprises at least one phosphatebackbone modification.
 3. The isolated stabilized D-typeoligodeoxynucleotide of claim 2, wherein the oligodeoxynucleotidecomprises at least one phosphorothioate base.
 4. The isolated stabilizedD-type oligodeoxynucleotide of claim 2 wherein the oligodeoxynucleotidecomprises at least one phosphodiester base.
 5. The stabilized D-typeoligodeoxynucleotide of claim 1, wherein the oligodeoxynucleotidecomprises between 18 and 30 nucleotides.
 6. The isolated stabilizedD-type oligodeoxynucleotide of claim 1, consisting of the nucleic acidsequence set forth as one of: 5′XXTGCGCCGATGCAGGGGGG3′; (SEQ ID NO: 18)5′XXTGCATCGACGCAGGGGGG3′; (SEQ ID NO: 19) 5′XXTGCGTCGGTGCAGGGGGG3′;(SEQ ID NO: 20) 5′GGTGCATCGTTGCAGGGGGG3′; (SEQ ID NO: 30) or 5′GGTGGATCGATCCAGGGGGG. (SEQ ID NO: 40)


7. The isolated stabilized D-type oligodeoxynucleotide of claim 6,further comprising at least one additional G in the 5′ far flankingregion.
 8. An oligodeoxynucleotide delivery complex comprising thestabilized D-type oligodeoxynucleotide of claim 1 and a targetingmoiety.
 9. The oligodeoxynucleotide delivery complex of claim 8, whereinthe targeting moiety is selected from the group consisting of acholesterol, a virosome, a liposome, a lipid, and a target cell specificbinding agent.
 10. The oligodeoxynucleotide delivery complex of claim 8,wherein the stabilized D-type oligodeoxynucleotide and the targetingmoiety are covalently linked.
 11. A composition comprising an effectiveamount of the stabilized D-type oligodeoxynucleotide of claim 1 and apharmacologically acceptable carrier.
 12. A method of stimulating a cellof the immune system, comprising contacting the cell with an effectiveamount of the stabilized D-type oligodeoxynucleotide of claim 1, therebystimulating the cell.
 13. The method of claim 12, wherein the cell is amonocyte, a natural killer cell, or a dendritic cell.
 14. A method ofinducing an immune response in a subject, comprising administering atherapeutically effective amount of the stabilized D-typeoligodeoxynucleotide of claim 1, thereby inducing an immune response.15. The method of claim 14, wherein the immune response comprises acell-mediated immune response.
 16. The method of claim 15, wherein theimmune response comprises a natural killer cell response or a dendriticcell response.
 17. The method of claim 14, wherein the immune responseis the production of a cytokine.
 18. The method of claim 17, wherein thecytokine is inducible protein 1-(IP-10), interleukin 10 (IL-10),interferon alpha (IFN-α) or interferon gamma (IFN-γ).
 19. The method ofclaim 14, comprising administering to the subject the stabilized D-typeoligodeoxynucleotide in combination with a vaccine, thereby enhancingthe efficacy of the vaccine.
 20. The method of claim 14, wherein thesubject has an autoimmune disorder.
 21. The method of claim 14, whereinthe subject has a deficiency of the immune system.